Thèses sur le sujet « Plant abiotic stresses »

Abiotic stresses cause extensive loss to agriculture production worldwide. Cowpea is an important legume crop grown widely in tropical and subtropical regions where high temperature, ultraviolet-B (UVB) radiation and drought are the common stress factors limiting production. Various vegetative, physiological, biochemical and reproductive plant attributes were assessed under a range of UVB radiation levels in Experiment I and in a combination with two doses of each carbon dioxide concentration [CO₂], temperature, and UVB radiation and their interactions in Experiment II by using six cowpea genotypes and sunlit plant growth chambers. The dynamics of photosynthesis and fluorescence processes were assessed in 15 cowpea genotypes under drought condition in Experiment III in pot-grown plants under sunlit conditions. A distinct response pattern was not observed in cowpea in response to UVB radiation form 0 to 15 kJ; however, plants grown under elevated UVB showed reduced photosynthesis resulting in shorter plants and produced smaller flowers and lower seed yield. Increased phenolic compounds appeared to be a defense response to UVB radiation. The growth enhancements observed by doubling of [CO₂] were not observed when plants were grown in combination with elevated UVB or temperature which also showed the most detrimental effects on plant growth and seed yield. Results form Experiment I and II revealed that cowpea reproductive traits were highly sensitive to abiotic stresses compared to the vegetative growth and development. A total stress response index (TSRI) technique, derived from all vegetative and reproductive parameters, was used to screen genotypes for their stress tolerance to UVB or combination of stresses. An increase in water use efficiency while maintaining higher rate of photosynthesis was an important drought tolerance mechanism in tolerant cowpea genotypes. Using principal component analysis technique, four groups of the genotypes were identified for their drought tolerance. Evaluating same genotypes across stress conditions revealed that no single genotype has the absolute tolerance characters to all stress conditions. The identified diversity for abiotic stress tolerance among cowpea genotypes and associated traits can be used to develop tolerant genotypes suitable for an agro-ecological niche though traditional breeding or genetic engineering methods.

Grapevine represents one of the major economic crop species on a worldwide scale, with a world production approaching 70 million of tons and a harvest area of over 7 million hectares. Amongst the 60 species within the Vitis genus, Vitis vinifera L. is the mostly used for the production of wine and distilled liquors. Before the devastation of European viticulture caused by of the introduction of phylloxera from North America, varieties of V. vinifera used commercially for wine production in Europe were traditionally grown on their own roots. Subsequently, the use of rootstocks from the pest’s origin was introduced to provide resistance to this and other deleterious diseases and to save the fate of European viticulture. Rootstocks have been bred from a number of Vitis species, especially V. berlandieri, V. riparia, and V. rupestris, and are known, in addition to the enhanced resistance to phylloxera and other pathogens, confer tolerance to abiotic stresses (e.g. drought, high salinity and Fe-deficiency), regulate the size of the scion, affected fruit development/ripening, contribute to fruit quality and can alter specific aspects of postharvest fruit quality of a scion. Results presented in this Ph.D thesis are a part of a larger multi-disciplinary project called SERRES (Selection of new grape rootstocks resistant to abiotic stresses through the development and validation of molecular markers) granted by Ager foundation. Selection of resistant rootstocks is crucial for the development of sustainable agricultural models and, at the same time, for inducing a balanced vegetative/productive ratio, a different ripening progression in grape berries and, as well as, differences in their global quality. Improving the knowledge about the molecular, biochemical and physiological bases of stress resistance is an absolute requirement for the selection of genotypes able to cope with stress conditions without any negative consequences on the vegetative growth and production of high quality grape. Drought has an enormous impact on crop production, indeed, it is one of the major factors limiting plant productivity and cause a severe yield reduction. Based on the global climate models, which predict an increase in the aridity in the next future, water deficit may became the major limiting factor. In this context rootstocks may play an important role in limiting crop loss by improving water use efficiency, potential for survival, growth capacity and scion adaptability to stress conditions. Water deficit leads to many morphological and physiological changes across a range of spatial and temporal scales, including reduced expansion of aerial organs, maintenance of root growth, decrease in transpiration and photosynthesis, accumulation of osmotic compounds and ions, activation of detoxifying processes and, in parallel, the transcriptional regulation of a large number of genes. Oxidative stress is related to the accumulation of reactive oxygen species, such as H2O2, O2-, -OH, 1O2, and NO. These ROS are responsible for most of the oxidative damages in biological systems and cellular components. Thus, a strict control of ROS levels, throughout the expression of genes coding for superoxide dismutases (SOD), catalase (CAT), ascorbate peroxidase and glutathione peroxidise ROS scavenging enzymes, is mandatory for plant survival and the cross-talk between ROS accumulation and redox state is integrating part of a fine homeostasis control that plays a pivotal role in the plant response to stresses. Recently, a biochemical and physiological study of the M4 [(V. vinifera x V. berlandieri) x V. berlandieri x cv Resseguier n.1] novel candidate genotype to be used as rootstock in grapevine was performed. This genotype, established from 1985 by the DiSAA research group operating at the Milan University, was selected for its high tolerance to water deficit (WS) and salt exposure (SS). In comparison with the 101.14 commercial genotype, M4 un-grafted plants subjected to water and salt stress showed a greater capacity to tolerate WS and SS maintaining photosynthetic activity also under severe stress conditions and accumulating, especially at the root level, osmotic compounds and ions. In the first part of this thesis were reported results obtained from a large scale whole transcriptome analyses (RNA-seq) performed on root (whole apparatus) and leaf tissues of 101.14 (drought susceptible) and M4 plants sampled in progressive drought (five time points). Physiological analyses were performed on treated (water-stress, WS) and control (well-watered, WW) plants over all the sampling. The multifactorial analysis , which was performed on mRNA -seq data concerning to both the analyzed tissues (leaf and root), allowed us to evaluate the relative weight of the genotype (R: 101.14 and M4), of the type of stress imposed (Treatment, T: WW and WS) and of the time point considered (P: T1-T4), and to identify Differentially expressed Genes (DEGs) that are affected in a specific way or the combined action of these factors (R:T, R:P, T:P and R:T:P). In WS root dataset, all considered components (R, T and P) were found to affect the higher number of genes in comparison to other dataset (WS leaf). A first general observation comparing results of the multifactorial analyses performed on leaves and roots is that in root tissue the “treatment” seems to be the main variable explaining differential gene expression depend on the kind treatment imposed, whereas in leaf tissue the weight of the genotype (rootstock) appear to be the highest. This observation is not surprising, considering that the root system is the first organ perceiving the water deprivation stress and the main one actively responding to it. In this case it’s clear the kind of treatment imposed represent the main variable influencing expression whereas the effect of the genotype is less determinant on differential expression of genes. RNA-seq data were used to performed a Differential Cluster Analysis (DCA), which is based upon comparison of correlation between genes expression of a “reference” and a “target” organism and allowed us to identify conserved and diverged co-expression patterns between related organisms. This analysis allowed us to compared the transcriptomic responses of M4 and 101.14 rootstocks. As concerns plant hormones, it was showed an induction of auxin, JAs and GAs related-genes at the beginning of the stress kinetic in M4 stressed roots, whereas a up-regulation of these transcripts in unstressed root was observed in 101.14. The most interesting metabolic category was the “Secondary metabolism” one because several DEGs belonging to these metabolisms were founded in both root and leaf upon WS, but with a strong specificity of DEGs expression among two considered organs. Indeed, upon WS, roots and leaves of the tolerant genotype M4 exhibit an higher induction of stilbenes (i.e. STS) and flavonoids (e.g. CHS, F3H, LDOX, FLS) biosynthetic genes, respectively. We hypothesized the role of these genes in the control and balance ROS levels, in addition to the others well known ROS scavengers. In presence of water stress, M4 rootstock may acts differential mechanisms in root and leaves which leads to the production of molecules, such as resveratrol and flavonoids and these events may be related to a secondary antioxidant system in this rootstock. The higher resistance of M4 rootstock to water stress, in comparison to what observed in 101.14, should be related to these events. In the second part, in order to evaluate the effects of the rootstocks on grape berry quality and development/ripening, an RNA-seq experiment on Cabernet Sauvignon (CS) grafted onto M4 and 1103 Paulsen rootstocks was carried out. Whole berries were collected from CS/1103P and CS/M4 bunches at 45, 59, 65 days after full bloom (DAFB), in correspondence to the end of lag phase. At this moment most of grape berries reached véraison, the other samples (separating skin and pulp) were collected at 72, 86 and 100 DAFB. On the basis of physical (volume and colour) and chemical (Soluble Solids Concentration, SSC) parameters, the two rootstocks seem to induce a different development and ripening pattern on CS berries. To identify the same developmental phases of berries collected from CS/1103P and CS/M4, the expression profile of genes involved in phenols, sugar and organic acids metabolisms were overlapped. This approach allowed to establish that the green phase occurred at 45 DAFB in both combinations, while véraison happened at 72 and 86 DAFB for CS/M4 and CS/1103P, respectively. An mRNA-seq and a microRNA-seq experiments were carried out on CS berries sampled at pre-véraison (45 DAFB), véraison (72 and 86 DAFB for M4 and 1103P, respectively) and traditional CS vintage date (100 DAFB). For the statistical analyses on RNA-seq data a pairwise comparisons between M4 and 1103P genotypes were accomplished at each time point and a large numbers of DEGs related to auxin metabolisms were identified with enrichment and clustering analysis. It is well known the important role of auxins on grape berry development, so, it was decided to focus our attention on this hormone and to performed a characterization of grape ARF and AUX/IAA gene families. Indeed, in another work presented in this thesis, we showed that an NAA treatment just before véraison caused delayed grape berry ripening at the transcriptional and physiological level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). Considering these results, the differences observed among CS/M4 and CS/1103P in grape berry development and ripening should be related to a different regulation of auxin metabolism. Indeed, all transcripts/miRNAs analyses performed (RNA-seq, microRNA-seq and qPCR) highlighted important differences in the auxin metabolism among the two scion/rootstock combination. Our data suggest an important involvement in the control of grape berry development/ripening of genes that are related, on one hand to auxin action (ARF and AUX/IAA) and, on the other hand, to homeostasis of this hormone through the expression of genes involved in conjugation (GH3) and transport (PIN and ABCB). In this context, also miRNA have an important role, especially by controlling ARF–related genes (e.g. miR160 and miR167). In the case of fruit ripening, auxin acted as a positive regulator of genes that control grape berry size (e.g. expansin-related genes) before the véraison stage; it was indeed observed the up-regulation at the pre-véraison stage, which was different for CS/M4 and CS/1103P, of transcripts that control auxin-responsive genes (e.g. VvARF8A and VvARF1A). The induction of genes that belonged to ARF family was paralleled by the expression of transcripts that control auxin level(e.g. VvGH3-1) and action (VvIAA9, VvIAA15A, VvIAA16), suggesting that an accurate regulation of auxin homeostasis in grape berries at these phases. Moreover, control of auxin levels in grape berry seems pass through other mechanisms which involved control of transport-related genes in the early (ABCBs) and late (PINs) phases of berry development. Taking into accounts that at commercial CS harvest, CS/M4 berries berries were showing differences in some processes ripening-related (e.g. flavonoids metabolism) and a different regulation of auxin metabolisms, when compared to those of CS/1103P, auxin seems to act as negative regulators on some genes related to grape berry ripening but its induction at the pre-véraison stage could be necessary to triggers other metabolism involved in ripening processes.
La vite (genere Vitis) rappresenta una delle principali specie coltivate su scala mondiale , con una produzione che si avvicina ai 70 milioni di tonnellate e una superficie coltivata di oltre 7 milioni di ettari . Tra le 60 specie all'interno del genere Vitis, Vitis vinifera L. è la più utilizzata per la produzione di vino e distillati. Prima della devastazione della viticoltura europea causata dall'introduzione del parassita fillossera dal Nord America, le varietà di V. vinifera usate per la produzione di vino in Europa non erano innestate. Successivamente, l'utilizzo di portinnesti di origine americana ha permesso di fornire una maggiore resistenza al parassita e ad altre malattie che stavano seriamente compromettendo la viticolture Europea. I portinnesti più usati commercialmente derivano da incroci di svariate specie di vite, tra cui V. berlandieri, V. riparia e V. rupestris, e, oltre a migliorare la resistenza alla fillossera e altri patogeni, conferiscono caratteristiche di tolleranza a stress abiotici (come siccità, elevata salinità e Fe-carenza), regolano la crescita dell’acino, contribuiscono alla maturazione e alla qualità dei frutti, possono alterare alcuni aspetti legati alla qualità in post-raccolta dell’acino. I risultati presentati in questa tesi di dottorato sono parte integrante di un progetto multi- disciplinare chiamato SERRES (selezione di nuovi portinnesti di vite resistenti a stress abiotici attraverso lo sviluppo e la validazione di marcatori molecolari) e finanziato dalla fondazione Ager. La selezione e la caratterizzazione di portainnesti che conferiscano un maggiore grado di tolleranza agli stress abiotici è essenziale per lo sviluppo di modelli agricoli sostenibili e, allo stesso tempo, per l’induzione di un rapporto equilibrato tra fase vegetativa e produttiva, una progressione diversa della maturazione dell’uva, così come, differenze a livello qualitativo. Migliorare la conoscenza delle basi molecolari, biochimiche e fisiologiche della resistenza allo stress è un requisito fondamentale per la selezione di genotipi in grado di far fronte alle condizioni di stress senza conseguenze negative su crescita vegetativa e produzione di uva ad alta qualità. Lo stress idrico ha un impatto enorme sulla produzione agricola, infatti, è uno dei principali fattori che limitano la produttività delle piante e causano una grave riduzione della resa. Sulla base dei modelli climatici globali, che prevedono un aumento delle aree aride nel prossimo futuro, la carenza idrica può diventare il principale fattore limitante per la coltivazione. In questo contesto, i portinnesti potrebbero assumere un ruolo importante nel limitare la perdita di raccolto migliorando l'efficienza dell'uso dell'acqua, il potenziale di sopravvivenza della pianta e la capacità di crescita del frutto in presenza di condizioni avverse come siccità ed elevata salinità del suolo (stress osmotici). Lo stress idrico porta a molti cambiamenti morfologici e fisiologici, tra cui ridotta espansione della parte aerea, limitazione della crescita radicale, diminuzione della traspirazione fogliare e dell’efficienza fotosintetica, accumulo di ioni e osmoliti, attivazione di processi di disintossicazione e parallelamente la regolazione a livello trascrizionale di un elevato numero di geni. In seguito allo stress idrico, si innesca uno stress secondario legato all’accumulo di specie reattive dell'ossigeno (ROS), quali H2O2, O2-, -OH, 1O2 e NO. Le ROS sono responsabili della maggior parte dei danni ossidativi nei sistemi biologici e nelle componenti cellulari. Un rigoroso controllo dei livelli delle ROS è obbligatorio per la sopravvivenza delle piante e il cross-talk tra l’accumulo di ROS lo stato redox è parte integrante di un preciso controllo omeostatico che gioca un ruolo fondamentale nella risposta agli stress. Le piante innescano svariati meccanismi di riduzione del livello di ROS (ROS-scavenging) volti all’induzione dell’espressione di geni che codificano per gli enzimi superossido dismutasi (SOD) , catalasi (CAT), ascorbato perossidasi e glutatione perossidasi. Recentemente è stato condotto uno studio di caratterizzazione a livello biochimico e fisiologico di M4 [(V. vinifera x V. Berlandieri) x V. berlandieri cv Resseguier n.1], un nuovo genotipo di vite candidato ad essere utilizzato come portinnesto. Questo genotipo, studiato dal 1985 dal gruppo di ricerca DiSAA dell'Università degli studi di Milano, è stato selezionato per la sua alta tolleranza allo stress idrico (WS) e salino (SS). Se confrontate con il genotipo commerciale 101.14, le piante di M4 sottoposte a deficit idrico hanno mostrato una maggiore capacità di tolleranza e una più elevata attività fotosintetica anche in condizioni di stress gravi. Nella prima parte di questa tesi sono stati osservati i risultati ottenuti da un’analisi trascrittomica condotta su larga scala (RNA -Seq), effettuata su foglie e radici dei portinnesti M4 e 101.14 campionati in condizioni di stress idrico progressivo (5 time-points). Le analisi fisiologiche sono state effettuate sulle piante trattate (deficit idrico, WS) e di controllo (irrigate, WW) lungo tutto il campionamento. L'analisi multifattoriale, che è stata condotta sui dati mRNA-Seq, ci ha permesso di valutare il peso di tre diverse componenti sulla risposta allo stress: genotipo ( R : 101.14 e M4 ), tipo di stress imposto (Trattamento, T : WW e WS) e time-point considerato ( P : T1 - T4 ). Con questa analisi stato inoltre possibile identificare i geni differenzialmente espressi (GDE) legati all’azione specifica o combinata di questi fattori (R:T , R:P , T:P e R:T:P). In WS radice si è sempre osservati un numero maggiore di GDE rispetto alla foglia. Una prima osservazione generale confrontando i risultati delle analisi multifattoriali eseguite su foglie e radici, è che nel tessuto radice il "trattamento" sembra essere la variabile che ha un impatto maggiore sull’espressione genica, mentre nel tessuto fogliare il peso del genotipo (portinnesto) sembra essere il più elevato. Questa osservazione non è sorprendente, considerato che il sistema radicale è il primo organo a percepire lo stress causato dalla carenza idrica e quello principale atto alla risposta. In questo caso è chiaro che il tipo di trattamento imposto rappresenta la variabile principale che influenza l’espressione genica mentre l'effetto del genotipo è meno determinante. Con i dati RNA-seq è stata eseguita una “Differential Cluster Analysis” (DCA), che si basa sul confronto delle correlazioni tra le espressioni dei trascritti di un organismo “reference” e di un “target”. Questa analisi ci ha permesso di identificare i pattern di co-espressione genica (T1-T4) conservati e pattern non-conservati tra M4 e 101.14. Per quanto riguarda gli ormoni vegetali, è stata osservata un’induzione dei geni legati ad auxine, jasmonati ed etilene nelle radici di M4 sottoposte a stress, mentre una sovra-regolazione degli stessi trascritti è stata osservata in 101.14. La categoria metabolica più interessante, emersa dall’analisi DCA, è quella legata ai metaboliti secondari. Infatti sono stati individuati diversi GDE legati a questa categoria sia in radice che in foglia di M4, indotti in condizioni di stress, ed è stata evidenziata una forte specificità di espressione tra i due tessuti. Infatti, in condizioni di carenza idrica, radici e foglie del genotipo tollerante M4 mostrano rispettivamente una maggiore induzione dei geni legati agli stilbeni (i.e. STS) e ai flavonoidi (e.g. CHS, F3H, LDOX, FLS). Il ruolo di questi geni potrebbe essere legato al controllo e al bilanciamento delle specie reattive dell’ossigeno (ROS), in aggiunta ai classici meccanismi di ROS-scaveging (meccanismi antiossidanti primari). In presenza di stress idrico, M4 potrebbe attuare meccanismi differenziali in radice e foglie che portano alla produzione di molecole, come resveratrolo e flavonoidi, correlate ad un sistema antiossidante secondario presente solo nel portinnesto più tollerante. La maggiore tolleranza allo stress idrico di M4, in confronto a quanto osservato in 101.14, potrebbe essere relativo a questi eventi. Nella seconda parte di questa tesi, è stato valutato l’effetto dei portinnesti M4 e 1103P su sviluppo, maturazione e qualità delle bacche di Cabernet Sauvignon (CS). Per questo esperimento sono stati campionati da piante di CS/M4 e CS/1103P acini interi a 45, 59 e 65 giorni dopo la piena fioritura (GDF). Successivamente la maggior parte delle bacche di CS/M4 avevano raggiunto l’invaiatura, si è quindi deciso di separare bucce e polpe per i campionamenti successivi, condotti a 72, 86 e 100 GDF. Sulla base dei parametri fisici (volume e colore) e chimici (solidi solubili totali, SSC), i due portinnesti hanno mostrato una diversa influenza sulla cinetica di sviluppo e maturazione delle bacche di CS. Per identificare le stesse fasi di sviluppo dei frutti raccolti da CS/1103P e CS/M4, è stato condotta un’analisi di espressione preliminare, mediante sistema real-time PCR, sui geni coinvolti nella biosintesi di fenoli, zuccheri e acidi organici. Questo approccio ha permesso di identificare la fase verde a 45 DAFB in entrambe le combinazioni d’innesto, mentre l’invaiatura è stata individuata a 72 e 86 DAFB rispettivamente per CS/M4 e CS/1103P. Le analisi mRNA-seq e micro-RNAseq sono state effettuate sulle bacche in fase di pre-invaiatura (45 GDF), invaiatura (72 GDF per CS/M4 e 86 GDF per CS/1103P) e epoca di raccolta tradizionale di CS (100 GDF). Le analisi statistiche sono state condotte sui dati RNA-seq confrontando il rapporto tra i dati di espressione di CS/M4 e CS/1103P ad ogni punto della cinetica e per entrambi i tessuti. Le analisi di “clusterizzazione” e di arricchimento hanno evidenziato la presenta di un elevato numero di GDE legati a metabolismi auxinici. Le auxine hanno un ruolo fondamentale durante lo sviluppo e sulla maturazione della bacca, si è quindi deciso di concentrare la nostra attenzione su questa classe ormonale e di eseguire una caratterizzazione e un’analsi filogenetica delle famiglie geniche ARF e AUX / IAA sul genoma di PN40024. Il ruolo delle auxine in questi processi è stato studiato anche in un altro un altro lavoro presentato in questa tesi, durante il quale è stato dimostrato che un trattamento sugli acini d’uva in fase di pre-invaiatura con l’auxina sintetica NAA causa un ritardo nella maturazione, che si manifesta a livello fisiologico e di espressione genica, parallelamente alle quali è stata osservata l’induzione di un elevato numero di trascritti atti a controllare l’omeostasi delle auxine. Le analisi condotte con il software HORMONOMETER hanno suggerito che il recupero omeostatico atto a portare i livelli dell’ormone a concentrazioni meno elevate è avvenuto a soli 7 giorni dal trattamento. Questa ipotesi è fortemente supportata dalla sovra-regolazione di geni coinvolti nella coniugazione (GH3 -like) e nell'azione ( IAA4 e IAA31 -like) delle auxine. Considerando questi risultati, le differenze osservate tra CS/M4 e CS/1103P durante lo sviluppo e la maturazione della bacca potrebbero essere collegate ad una diversa regolazione dell’auxina. Infatti, i dati di espressione (mRNA-seq, microRNA-seq e qPCR) evidenziato importanti differenze nel metabolismo auxinico tra le due combinazioni d’innesto. I nostri dati suggeriscono un coinvolgimento importante dell’ormone nel controllo dello sviluppo/maturazione della bacca grazie all’espressione di legati, da un lato all’azione delle auxine (ARF e AUX/IAA) e, dall'altro , all’omeostasi di questo ormone attraverso trascritti coinvolti nella coniugazione (GH3) e nel trasporto (PIN e ABCB). In questo contesto , anche i miRNA hanno un ruolo importante, in particolare esercitando un controllo sulla trascrizione dei geni ARF (e.g. miR160 e miR167). In fase di pre-invaiatura, le auxine hanno un’azione positiva sulla trascrizione dei geni che controllano le dimensioni della bacca (e.g. espansine) e di geni legati alla famiglia delle ARF (ad esempio VvARF8A e VvARF1A ). Parallelamente all'induzione di geni che appartengono alla famiglia ARF, è stata osservata l’induzione di trascritti che controllano i livelli (e.g. VvGH3-1) e l'azione (VvIAA9, VvIAA15A, VvIAA16) dell’ormone, suggerendo un’accurata regolazione dei livelli auxinici in queste fasi importanti dello sviluppo del frutto. Inoltre, il controllo dei livelli di auxina nella bacca d’uva sembra essere legato anche ad altri meccanismi legati all’induzione di geni legati al trasporto ormonale durante le fasi precoci (ABCBs) e tardive (PIN) della maturazione del frutto. Tenendo conto delle differenze osservate tra CS/M4 e CS/1103P nell’espressione di trascritti legati al metabolismo dell’auxina, questo ormone sembra esercitare un’azione negativa su alcuni geni legati alla maturazione della bacca (e.g. flavonoidi), ma la sua induzione nella fase di pre-invaiatura potrebbe essere necessaria per far scattare altri processi metabolici coinvolti nella maturazione dell’acino d’uva.

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

Why don’t crop plants grow as fast as they should? In optimal conditions, elite crop varieties routinely outperform those grown in the average field. The vast majority of this reduction in growth activity is due to abiotic stresses such as drought, heat, and nutrient limitation. Abiotic stress reduces plant growth by triggering a reduction of meristem size and causing premature differentiation of proliferating cells. Differentiated cells are no longer able to divide, and smaller meristems have a reduced capacity to restore growth when the abiotic stress passes. We have designed and evaluated a novel high-throughput screening system to identify compounds able to reduce or prevent this premature differentiation in order to retain modest growth capacity in stressful conditions and enable rapid recovery from stress. Such chemicals can be applied to crop plants using existing agricultural methods, and because there is no need for genetic modification, it is widely applicable to many different crop species. Using the novel technique of flow sorting followed by protoplast culture, we have developed a high-throughput automated confocal imaging method to screen chemicals for their effects upon cell differentiation. Meristem protoplasts isolated from the root tips of pROW1:GFP Arabidopsis plants were monitored for differentiation when exposed to different chemicals. To evaluate this system, a library of biologically active small molecules provided by Syngenta was screened against protoplasts and whole plants. Several compounds were identified with the ability to improve Arabidopsis root growth in in vitro growth conditions. Two subsets of these chemicals were identified: a subset of chemicals that improved stress tolerance through modulation of post-meristem differentiation, and a subset of chemicals that improve growth rate by increasing rates of cell division in the root apical meristem. This screening system is able to detect the subset of chemicals that was shown to affect postmeristem differentiation, but not the other subset. No false positives were detected. These results suggest that this single-cell screening system is a powerful, high-throughput method suitable for the detection of molecules for use in crop protection.

Abiotic stresses are responsible for limiting crop production worldwide. Among diverse abiotic stresses, drought and salinity are the most challenging. Plants under these conditions have diverse strategies for tolerating stress. Osmotic adjustment and osmoprotection occur in plants during salinity and drought stress through accumulation of compatible solutes to a high level without interfering with cellular metabolism. Polyols (sugar alcohols) including sorbitol and ribitol are one such class of compatible solutes. Using plants of wild-type (WT) and three genetically-modified lines of tomato (Solanum lycopersicum cv. ‘Ailsa Craig’), an empty vector line ‘TR22’, and 2 sdh anti-sense lines ‘TR45’, and ‘TR49’ designed to severely limit sorbitol metabolism, the objective of this work was to characterize the sorbitol cycle in tomato in response to abiotic stresses. Sorbitol and ribitol content, as well as the enzymatic activities, protein accumulation, and gene expression patterns of the key sorbitol cycle enzymes ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), ALDOSE REDUCTASE (AR), and SORBITOL DEHYDROGENASE (SDH), were measured in mature leaves in response to drought stress by withholding water and by using polyethylene glycol as a root incubation solution to mimic drought stress, to salt stress by incubating roots in NaCl solution, and to incubation of roots in 100 mM sorbitol and ribitol. A6PR, not previously reported for tomato, and AR both exhibited increased activity correlated to sorbitol accumulation during the drought osmotic, and salt stresses, with SDH also increasing in WT and TR22 to metabolize sorbitol. The level of sorbitol accumulation was considerably lower than that of the common sugars glucose and fructose so was not enough to have a significant impact on tissue osmotic potential but could provide other important osmoprotective effects. Use of the sdh antisense lines indicated that SDH has the key role in sorbitol metabolism in tomato as well as a likely role in ribitol metabolism. Like sorbitol, ribitol also accumulated significantly more in the antisense lines during the stresses. Expression and/or activity of A6PR, AR, and SDH were also induced by the polyols, although it is not clear if the induction was due to a polyol signal, the osmotic effect of the incubation solution, or both. In addition, a unique post-abiotic stress phenotype was observed in the sdh anti-sense lines. After both drought and salt stresses and during a recovery phase after re-watering, the antisense lines failed to recover. This may have been due to their accumulation of ribitol. The sdh anti-sense lines were uniquely sensitive to ribitol but not sorbitol, with an apparent foliar and seed germination toxicity to ribitol. The determination that sorbitol, and perhaps ribitol as well, plays a role in abiotic responses in tomato provides a cornerstone for future studies examining how they impact tomato tolerance to abiotic stresses, and if their alteration could improve stress tolerance.

Achillea collina Becker ex Rchb., a medicinal plant rich in volatile compounds, was used to study the effects of biotic and abiotic stresses over plant growth and secondary metabolism. Biotic stress was induced by Myzus persiceae Sulzer and Macrosiphoniella millefolii (De Geer ), a generalist and specialist aphid species respectively. Abiotic stress was caused by mechanical damages provoked by a pin and a specially built equipment which apply a controlled and extended pressure to the plants. Plant growth and volatile compounds emissions were evaluated in the different experimental conditions analyzed. The effect of jasmonic acid on the plant volatile fingerprint was also evaluated. The volatile emission patterns obtained in the different conditions were compared in order to have suggestions regarding the metabolic pathways activated in each situation. Furthermore pea (Pisum sativum L.) and peach (Prunus persica L. Batsch) volatile fingerprints due to M. persicae infestation were analyzed and compared to those obtained from A. collina. The comparison of the results lead to the identification of volatile compounds induced only by the aphids in all the plant species studied, suggesting the activation of a common metabolic pathway due to infestation. Preliminary molecular approach seems to confirm pytochemical data.

Bacterial endophytes are the organisms that live inside the plant for a full or a part of their life cycle. Endophytic bacteria have captured the interest of agriculture industry due to their plant beneficial properties, such as synthesis of phytohormones, solubilization of soil nutrients, and alleviation of biotic and abiotic stresses. Several studies have reported that stress tolerant endophytic bacteria can work with a similar performance as non-stressed conditions when inoculated to the plants under stressed conditions. Combination of abiotic stresses such as salinity, drought and low nitrogen stress can have additive or agonistic effects on bacterial and plant growth, and their interactions. However, very few studies have reported the impact of combined stress on endophytic bacterial assisted plant growth promotion. Therefore, understanding the underlying mechanisms of endophytic bacterial assisted plant’s tolerance abiotic stresses may provide the means of better exploiting the beneficial abilities of endophytic bacteria in agricultural production. Thus, the aim of this thesis was to study the stress tolerance mechanisms, beneficial characteristics, and plant growth promotion characteristics of endophytic bacteria under individual and combined abiotic stresses. Transcriptome analysis of endophytic bacteria revealed that tolerance mechanisms to deal with one kind of stress is different than concurrent stresses. Salinity and drought stress largely modulated the genes involved in flagellar assembly and membrane transport, showing reduced motility under stress conditions to preserve the energy. Additionally, bacterial endophyte that can fix nitrogen was studied with maize plant growth promotion under drought and low nitrogen stress conditions. The results suggested that diazotrophic bacterial endophyte can promote plant growth under moderate individual and combined stress conditions. Plant growth promoting endophytic bacteria can be utilized as an efficient tool to increase crop production under individual and concurrent abiotic stresses.

Les herbicides sont des polluants suscitant de grandes inquiétudes en raison de leur ubiquité environnementale résultant de leur usage intensif dans l’agriculture moderne et de leur persistance dans les sols et les eaux. Les herbicides peuvent être dégradés par des microorganismes, des plantes ou d’autres processus naturels, produisant alors une vaste gamme de métabolites dont l’impact sur les écosystèmes reste méconnu. Dans un contexte d’évaluation des risques environnementaux, l’étude de la réponse des plantes à des mélanges complexes de xénobiotiques est importante pour estimer les effets des contaminations, notamment dans le cas de pollution résiduelle. Afin d’étudier l’impact de cette diversité de polluants, les mécanismes de réponse et les cibles impliquées, la plante modèle Arabidopsis thaliana a été confrontée à des doses variables de molécules de la famille des triazines constituant une série chimique cohérente : atrazine, herbicide encore largement utilisé au niveau mondial, déséthylatrazine, métabolite chloré de l’atrazine, et hydroxyatrazine, métabolite de déchloration de l’atrazine. Ce travail montre que l’exposition de courte durée à des doses variables d’atrazine, de déséthylatrazine et d’hydroxyatrazine, au niveau racinaire, affecte de manière spécifique et dose-dépendante la croissance précoce et le développement de la plante. La caractérisation d’effets directs et multiples sur la respiration et la croissance racinaire a permis de révéler des mécanismes d’action non-canoniques, distincts de l’action classiquement décrite des triazines sur le photosystème II. Afin d’identifier ces mécanismes, activés en absence de dommages cellulaires, une analyse transcriptomique au niveau du génome entier a été effectuée. Les trois triazines induisent des changements coordonnés et spécifiques dans l’expression des gènes. L’analyse fonctionnelle des gènes différentiellement exprimés et de leur promoteur révèle que les voies de signalisation liées à la fois aux hormones végétales, à la perception de faibles niveaux d’énergie, aux stress environnementaux ainsi qu’aux interactions biotiques sont impliquées dans la réponse aux faibles doses de triazines. Les triazines affectent, en particulier, l’expression de gènes connus pour être régulés par les cytokinines. De manière intéressante, cette famille d’hormones végétales montre des caractéristiques chimiques similaires à celles des triazines. Des études développementales utilisant différentes modalités d’exposition aux triazines et aux cytokinines ont alors été effectuées sur des génotypes sauvages et sur des mutants de la voie de signalisation des cytokinines. L’identification d’interactions spécifiques entre les triazines et les composants de la signalisation des cytokinines a alors mis en évidence des mécanismes potentiels de compétition et/ou d’antagonisme. La caractérisation de ces perturbations au niveau de la transduction du signal pourra permettre à terme d’évaluer l’efficacité des herbicides sur les cultures ainsi que l’impact des contaminations xénobiotiques sur les communautés végétales naturelles. Enfin, l’identification des interactions entre stress xénobiotique, biotique et abiotique approfondira les connaissances sur les effets croisés de la pollution chimique et des stress liés au changement climatique
Herbicides are pollutants of high concern due to their environmental ubiquity resulting from extensive use in modern agriculture and persistence in soil and water. Degradation events on active molecules mediated by microorganisms, by plants and by natural processes give rise to a plethora of herbicide metabolites of unknown impact on ecosystems. Study of plant behavior toward such complex mixtures of xenobiotic structures is important to evaluate the effects of contaminations, especially in the context of residual pollution. In order to understand the mechanisms underlying the action of this diversity of compounds, the model plant Arabidopsis thaliana was confronted to variable doses of the widely-used triazine herbicide atrazine, and of two of its metabolites, desethylatrazine and hydroxyatrazine. Short exposure to varying concentrations of atrazine, desethylatrazine and hydroxyatrazine was found to affect early growth and development in various dose-dependent and distinct manners. These differential effects pointed out to the multiple involvement of non-canonical mechanisms, directly affecting respiration and root development. In order to identify these mechanisms, which are activated in the absence of major adverse physiological effects, a genome-wide transcriptomic analysis was carried out. All of the triazines under study induced coordinated and specific changes in gene expression. Functional analysis of differentially expressed genes and of their promoters revealed that signaling pathways related to plant hormones, low energy sensing, environmental stresses and biotic interactions were involved in low-dose triazine responses. In particular, triazines affected the expression of genes known to be regulated by cytokinins. Interestingly, this family of plant hormones shares similar chemical features with triazine compounds. Developmental studies on plants bearing mutations in cytokinin sensing and signaling pathways were then carried out under variable triazine exposures. The identification of specific interactions between triazine compounds and cytokinin-signaling components highlighted potential mechanisms of competition and/or antagonism. The characterization of such signal transduction modifications and perturbations will be useful to assess herbicide efficiency in crop systems and xenobiotic contamination impact on natural plant communities. Finally, the identification of crosstalk processes between xenobiotic, abiotic and biotic stress signaling gives novel insights into the interplay between chemical pollution and climate change stressors

Medicinal plants have always been considered a rich source of secondary metabolites that promote human health. Quality and property of medicinal plants strictly depend on secondary metabolites profile. They also play important roles in plant physiological processes and in ecological systems. The environment exerts a selective pressure on plants and these molecules actively participate to the plant response and adaptation. Amongst secondary metabolite, the phenolic compounds possess properties able to prevent oxidative stress. Therefore, an enhancement of the amount of phenolic compounds can be observed under different environmental factors. With this project we aimed to study the phenolic compounds of the medicinal plant Achillea collina Becker ex Rchb. cv “SPAK”, and their implication in physiological and biochemical response to abiotic and biotic stresses. We seek the possibility to increase the synthesis of phenolics with health properties or useful as potential control agents of insect pests. Abiotic stress. Hydroponic culture was used to evaluate the effect of long-term mineral, nitrogen starvation (abiotic stress) in A. collina. By means of HPLC-DAD-ESI/MS and NMR techniques, the content and the qualitative profile of A. collina methanol soluble phenolics, were evaluated. We concluded that the methanol extracts of A. collina leaves and roots are rich in hydroxycinnamic acids such as chlorogenic acid (2.33 ± 0.3 mg g-1 Dw), 3,5-di-O-caffeoylquinic acid (10.7 ± 4.2 mg g-1 Dw) and 4,5-di-O-caffeoylquinic acid (0.88 ± 0.24 mg g-1 Dw). The content of hydroxycinnamic acids significantly increased in plants growth under mineral nitrogen starvation, respect to the control plants. Chlorogenic acid increased by 2.5 and 3-fold and 3,5-di-O-caffeoylquinic acid increased by 8.5 and 35-fold in leaves and root, respectively. Biotic stress. A. collina plants cultivated in soil were infested with the phloem feeders aphids. We set up the system (e.g., age of plant, type of the cage, number of insects per plant, duration of infestation) to co-cultivated the plants with specialist (Macrosiphoniella millefolii) and generalist (Myzus persicae Sulzer) aphids. Plant growth, water and total protein content were evaluated. Based on a preliminary assessment of phenolic fingerprint, further extractions and separations were performed on A. collina leaves, to obtained soluble and cell wall-bound fractions and their sub-classes. Our results showed that A. collina plants were strongly affected by aphid infestation. Twenty days after infestation, the fresh weight was twenty-fold and seven-fold increased, in control and infested plants. Water and protein content, condensed tannins and methanol soluble phenolics content, were not affected by the aphid infestation. Cell wall-bound phenolics content increased in infested plants. The main phenolics were found to be chlorogenic acid and 3,5-di-O-caffeoylquinic in methanol soluble fraction, and caffeic acid in cell wall fraction. The chromatographic profiles showed that the main hydroxycinnamic acids were present in control and in both M. persicae and M. millefolli infested plants. The quantitative analysis indicated that the levels of chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were 44% and 37% higher in M. persicae infested plants, respectively. The levels of chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were 27% and 39% higher in M. millefolli infested plants, respectively. Twenty days after infestation the content of caffeic acid was resulted 43% and 34% higher in M. persicae and M. millefolli infested plants, respectively. These differences should indicate the different evolutionary interaction between plant and generalist/specialist aphid. We hypotheses that the increase of these molecules may represent a plant resistance mechanism against aphid attack. Finally, a chemometric approach, by means multivariate statistical analysis, was applied on chromatogram profiles to verify whether there is difference between methanol soluble fraction of infested and non infested A. collina plants. The discriminant analysis showed a significant effect of phloem feeders aphids on soluble phenolic compounds and indicated two peaks, not yet identified, that separate control from infested plants. In conclusion the model system developed to cultivate A. collina was useful to understand the metabolic basis of the environment interactions. The main hydroxycinnamic acids identified, were resulted increased in both abiotic and biotic stress, suggesting their implication in A. collina protection to environmental controversies.

Plants growing in the field are affected by several adverse environmental conditions at the same time. The simultaneity of abiotic factors affecting plants creates a new stress situation different from the individual ones. Global warming is increasing earth surface temperature, and this is accompanied by other environmental changes (soil degradation, increase of drought periods, changes in precipitation pattern). All these changes will affect the arable lands around the world and crop production will be reduced unless new cultivars capable to face the environmental changes are developed. Therefore, the main objective of this PhD is to study the impact of different abiotic stresses in combination with high temperatures on plant physiology. To achieve this objective, this work has focus in studying physiological, hormonal and transcriptomic responses to different stress situations in citrus and model plants to obtain a global view of plant mechanisms to tolerate complex abiotic stress situations.
Las plantas cultivadas se ven afectadas por numerosas condiciones ambientales adversas al mismo tiempo. Cuando dos o más factores abióticos afectan a las plantas de manera simultánea se crea una nueva condición de estrés, diferente a la provocada por los estreses individuales. La temperatura de la superficie terrestre está aumentando, y esto viene acompañado por otros cambios medioambientales. Todos estos cambios afectan a las zonas de cultivos de todo el mundo y a la producción agrícola. El objetivo principal de esta tesis es estudiar el impacto en la fisiología de las plantas de diversos estreses abióticos en combinación con las altas temperaturas. Para conseguir este propósito, este trabajo se ha enfocado en el estudio de las respuestas fisiológicas, hormonales y transcriptómicas de las plantas a diferentes condiciones de estrés para obtener una visión global del mecanismo de tolerancia de las plantas a situaciones complejas de estrés abiótico.

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs. MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation. Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life.

In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented: - Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS). - Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays. - Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches.
Las proteínas LEA, originalmente fueron descritas en las semillas de algodón; se acumulan en grandes cantidades en estructuras tolerantes a la desecación (semillas, polen) y en tejidos vegetativos sometidos a estrés abiótico, sequía, salinidad y frío. También se hallan en organismos anidrobióticos, en plantas de resurrección, algunos invertebrados y microorganismos. La presencia de proteínas LEA se correlaciona con la adquisición de tolerancia a la desecación. Desde un principio se les atribuyó un papel en las respuestas de las plantas en la adaptación al estrés (revisado en Bartels and Salamini 2001, Tunnacliffe 2007, Shih et al. 2010, Tunnacliffe 2010, Hand et al. 2011). Las proteínas LEA se clasifican en diversos grupos en función de dominios y secuencias de aminoácidos específicos (Wise 2010, Batagglia et al 2008, Bies-Ethève et al 2008). Los grupos 1, 2 y 3 son los más relevantes ya que abarcan la mayoría de las proteínas de la familia LEA. Una característica general de estas proteínas es su elevada hidrofilicidad, alto contenido de aminoácidos cargados y su falta de estructura en estado hidratado. A pesar de encontrarse mayoritariamente en forma de “random coil”, algunas adquieren un cierto grado de estructura durante la deshidratación o en la presencia de agentes promotores de α-hélices (Shih et al. 2010, Hand et al. 2011). A nivel celular se han hallado en todas las localizaciones, citosol, núcleo, nucleolo, mitocondria, cloroplasto, vacuola, retículo endoplásmico, peroxisoma y membrana plasmática, donde se supone ejercen su función protectora frente al estrés (Tunnacliffe and Wise 2007, Hundertmark and Hincha 2008). En relación a las modificaciones post-traduccionales, algunas se hallan fosforiladas (Jiang and Wang 2004; Plana et al. 1991, Heyen et al. 2002, Rohrig et al. 2006). Los efectos protectores de las varias proteínas LEA se han demostrado mediante ensayos in vitro y en aproximaciones transgénicas que han dado lugar a fenotipos resistentes a la sequía, sal y frío. Por lo general, se considera que estas proteínas contribuyen a la protección y a la estabilización de macromoléculas y estructuras celulares en las respuestas de adaptación al estrés en plantas; sin embargo, sus funciones específicas aún no han sido esclarecidas. A nivel molecular se ha propuesto que las funciones de las proteínas LEA pueden ser variadas: estabilización y renaturalización de proteínas, mantenimiento de membranas, en combinación, o no, con azúcares, tampones de hidratación (substitución de moléculas de agua), afinidad por iones y función antioxidante (Tunnacliffe and Wise 2007, Shih et al. 2010, Batagglia et al. 2008). Para finalizar, diremos que los objetivos principales de esta tesis consisten en ampliar los conocimientos sobre las proteínas LEA y sus funciones relativas a la tolerancia a la sequía. Los resultados están presentados en forma de capítulos.

Most plants complete their life cycle in a single location and therefore are affected by the changing environment. As a result, plants have evolved physiological and developmental adaptations to overcome stress. The work presented in this thesis has examined the regulation of photosynthetic electron transport in barley, rice and Thellungiella salsuginea. Barley is considered as a crop which is comparatively tolerant to soil salinity. The focus of this study was to evaluate the physiological responses of photosynthesis in barley under salinity and to characterize traits responsible for the regulation of photosynthesis. At low salt concentrations, barley plants protect PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH, by cyclic electron transport associated with PSI, to support nonphotochemical quenching (NPQ). However, at the highest concentration of salt examined, this regulation starts to fail. The failure might result from a specific loss of PSI, resulting in reduced cyclic electron flow, or an increase in the leakiness of the thylakoid membranes, resulting in loss of ΔpH. The effects of salinity on the regulation of electron transport through Photosystem I and Photosystem II have been studied in two rice varieties from Sri Lanka. The regulation of photosynthesis in the salt-tolerant At-354 is more prominent than in the salt-sensitive Bg-352 when plants are exposed to salt. Exposure of Bg-352 to salt resulted in a substantial decrease in gas exchange, PSII photochemistry, leaf area and loss of chlorophylls. The decrease in the photosynthesis in AT-354 is caused by stomatal limitations, which restrict the CO2 entry into the plants, whereas the decrease of photosynthesis in Bg-352 is caused by non-stomatal limitations. Results suggest that At-354 protects PSII centres from excitation pressure by down-regulating the electron transport chain and maintaining ΔpH by cyclic electron transport associated with PSI to support NPQ. At high salt concentration, this regulation starts to fail in Bg-352.Tolerance to abiotic and biotic stress has evolved in many wild plant species, termed extremophiles. These plants contain essential genes which may used to improve crop production in changing environments. Thellungiella salsuginea is an extremophile, able to grow and reproduce in extreme environments. Stepien and Johnson (2009) identified a protein, known as the plastid terminal oxidase (PTOX) which acts as an alternative electron sink in T. salsuginea under salt stress. The current study showed that, in addition to salt, T. salsuginea showed increases in PTOX protein content and activity when exposed to drought, different growth irradiances and cold with high light. Semi-natural conditions also triggered the activity of PTOX. This study also showed that salt caused an up-regulation of PTOX gene transcripts in the leaves of salt treated T. salsuginea plants compared to control plants. Direct electron transport from PSII to PTOX and then to oxygen via the PQ pool accounted for up to 30% of total PSII electron flow in T. salsuginea (Stepien and Johnson, 2009). Efficient electron flow from PSII to PTOX would however, probably require co-location of these complexes in the same thylakoid fraction. To examine the location of PTOX in the thylakoid membrane, immunoblot analyses were performed, to test for changes in other protein complexes which may be associated with PTOX. In addition blue-native polyacrylamide gel electrophoresis and immunoblots were performed to isolate and detect the PTOX protein with any associated complexes. Although immunoblot analysis showed a prominent signal, mass spectrometry data did not allow identification of PTOX. This results suggests that further studies are needed to identify the precise localisation of the PTOX protein in the thylakoid membranes in T. salsuginea.

Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes.
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Plants respond to environmental stress by altering their gene expression. Under stress conditions some genes are activated and some genes are repressed. Even though a lot of work has been done to understand mechanisms of gene activation under abiotic stress very little information is available on how stress responsive genes are kept repressed under normal growth conditions. Recent work has revealed that plants use transcriptional repression as common mechanism of gene repression. Transcriptional repression is achieved by recruitment co-repressor complexes to the target genes. Recent studies have revealed that the co-repressor LUH complexes with SLK1 and SLK2 to silence Arabidopsis thaliana stress responsive genes. However, the transcription factors involved in the recruitment of this complex to its target genes are not known. In this study, we identified SLK2INT1, as a novel transcription factor that is involved in silencing of select Arabidopsis thaliana stress responsive genes by recruiting the LUH-SLK2 complex.

Bambara groundnut (Vigna subterranea (L.) Verdc) is an indigenous legume that is still cultivated in subsistence agricultural systems in sub-Saharan Africa, despite the lack of any major research effort until recently. The crop is cultivated from local landraces as there are no true varieties of the species bred for specific traits. The variable and hostile climates in the region mean that annual yields of most rainfed crops including bambara groundnut are far below their agronomic or genetic potential. The lack of quantitative information on the eco-physiological responses of the crop to various abiotic factors has resulted in poor decision making on crop management practices especially in relation to sowing date and the selection of appropriate landraces for different locations. Modelling of bambara groundnut was initiated previously but there is still insufficient understanding of how growth and developmental processes can be simulated under abiotic stress and different photoperiods. The aim of this study was to develop a crop simulation model for bambara groundnut to predict growth, development and yield under drought, heat and cold stress and different daylengths. The present model (BAMGRO) is an adaptation of the established CROPGRO and previous bambara groundnut models; BAMnut and BAMFOOD project model. It uses climate data, landrace specific parameters and soil characteristics and runs on a daily time-step to determine the canopy development, biomass production and yield of a landrace in a specific environment. The parameters of the model have been determined with glasshouses data (TCRU, University of Nottingham) and published information. BAMGRO is capable of describing differences between landraces, and the influence of drought, temperature and photoperiod using a simplified approach. The present modelling approaches with BAMGRO model provide useful predictive information on canopy development, biomass production and yield formation of bambara groundnut landraces under contrasting environments. Two contrasting landraces; Uniswa Red (Swaziland) and S19-3 (Namibia) were used in the present study to evaluate the growth and yield performances under drought, heat and cold stress. BAMGRO has been primarily validated against independent data sets of two years glasshouse for two contrasting landraces; Uniswa Red and S19-3 grown under two temperatures (23 ± 5 0C, 33 ± 5 0C) with drought. Further, it was validated for field data in Botswana with two sowing dates (January 18, February 1) during the 2007 season and for Swaziland for three landraces; Uniswa Red, DipC, OM1. The model achieves a good fit between observed and predicted data for LAI (Nash and Sutcliffe (N-S), 0.78-0.98; Mean Absolute Error, ± 0.14-0.57) for tested four landraces. Pod yield simulation was correlated well with measured values especially for Uniswa Red and S19-3 (N-S 0.73-0.87; Mean Absolute Error ± 16 g m-2) while it was poor for DipC and OM1 (N-S, 0.46-0.50; Mean Absolute Error, ± 15.6-17.7 g m-2). Further, the comparison of simulated and measured data of TDM reported lower correlation compared to LAI and yield. (N-S, 0.59-0.79; Mean Absolute Error ± 48-100 g m-2) indicating overall underestimation. The performance of the BAMGRO-soil water module was tested by validating the available soil moisture and results indicating that it over estimated for upper layers while deeper layers showed lower prediction. The possible reasons for the discrepancies in measured and simulated data are differences in quality and quantity of solar radiation in UK summer and Semi-arid Africa, intra-landrace variability and poor calibration of soil water module. Four potential applications of BAMGRO and three future developments are presented in this thesis.

Polyploidy, or genome doubling, is an important process in plant evolution that has effects on phenotypes, such as changes in flowering time. Allopolyploidy can result in considerable genetic changes including alterations to genome structure, DNA methylation patterns, and gene expression. Although the expression of duplicated genes in polyploid plants has been extensively studied, little is known about the effects of abiotic stress conditions on homeologous gene expression. In this thesis, I examined the expression of 30 homeologous gene pairs in response to five abiotic stress treatments, using a single strand conformation polymorphism (SSCP) assay in allotetraploid Gossypium hirsutum. Twenty-two genes showed stress-induced changes in the expression ratio of the two homeologs, and eight genes showed reciprocal expression changes in response to different abiotic stress treatments, suggesting quantitative subfunctionalization. I also examined the expression of ten homeologous gene pairs in response to three abiotic stress treatments in a synthetic Gossypium allotetraploid. Eight genes showed stress-induced expression changes. Comparison of the expression changes showed that there was little correspondence in the stress-induced homeolog expression patterns between the natural and synthetic Gossypium polyploids. The results of this study indicate that abiotic stress conditions can have considerable effects on expression of homeologous genes. Some of those expression changes might help plants survive abiotic stresses.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O H2O2 à tido como molÃcula sinalizadora de vÃrios eventos celulares e seu nÃvel endÃgeno à controlado pelas diferentes isoformas de APX e CAT que estÃo distribuÃdas na cÃlula vegetal. No presente estudo, foram utilizadas diferentes abordagens visando um melhor entendimento do papel das isoformas citosÃlicas de APX, consideradas por muitos autores as isoformas mais importantes dentre as APXs, e sua sincronia com CAT, tendo em vista que estas sÃo as principais removedoras do H2O2 intracelular. Inicialmente, foi observado que plantas de arroz submetido à combinaÃÃo de estresse salino e alta temperatura apresentaram modulaÃÃo positiva da atividade de APX associada com menor acÃmulo de H2O2. Intrigantemente, plantas duplamente silenciadas nas isoformas de APX citosÃlica (APx1/2s) apresentaram desenvolvimento normal em condiÃÃes controle. Em condiÃÃes estressantes, as APx1/2s exibiram um mecanismo antioxidativo compensatÃrio mediado principalmente pelas isoformas de GPX citosÃlica e cloroplÃstica juntamente com antioxidantes nÃo enzimÃticos. AlÃm disso, as APx1/2s submetidas a inibiÃÃo irreversÃvel de CAT apresentaram melhor desempenho fotossintÃtico e menores danos oxidativos que plantas que continham a APX citosÃlica, CAT ou ambas indicando que outros mecanismos compensatÃrios foram ativados para mitigar os possÃveis efeitos negativos do acÃmulo de H2O2. Tomados em conjunto, os dados indicam que as isoformas citosÃlicas de APX nÃo sÃo as enzimas mais importantes no controle dos nÃveis de H2O2, visto que plantas com ausÃncia destas isoformas completam o ciclo de vida e sÃo capazes de enfrentar situaÃÃes de estresse. AlÃm disso, à possÃvel afirmar que a aÃÃo coordenada das isoformas de GPX, juntamente com ascorbato e glutationa reduzidos, podem ser os responsÃveis pela manutenÃÃo do equilÃbrio redox celular em condiÃÃes estressantes em plantas de arroz, diferente do que à noticiado para a planta-modelo Arabdopsis.
Plants in the field are exposed to multiple stresses, such as salinity, drought, high light, heat/cold and others, resulting in different physiological responses. T o evaluate the consequences of some of these stresses to the photosynthetic apparatus and an tioxidative metabolism, 30 day old rice plants , were submitted to the following treatments: control (without NaCl and at 27 ÂC), heat stress (without NaCl and at 42 ÂC), salt stress (with 100 mM NaCl and at 27 ÂC) and combined stress (salt+heat). The contr ol and salt stress treatments lasted 8 days and the heat and combined stress treatments lasted 6 hours. At the end of the experimental period, gas exchange, chlorophyll fluorescence and electrolyte leakage were measured and the leaves were collected for bi ochemical determinations. I solated salt and heat stress es were not sufficient to cause damage in the photochemical apparatus and heat stress only modifie d stomatal aperture. In combined heat and salt stress , the results indicate that photosynthetic process es were affected at the level of CO 2 assimilation and quantum efficiency. E lectrolyte leakage, TBARS and H 2 O 2 content were elevated in sal t +heat treatment, but in isolate d heat stress the TBARS was decreased. Reduced ascorbate and glutathione were similarly decrease d in plants exposed to the combination of salt and heat. A ll enzymes examin ed here were differently modulated in experimental treatment s . Taken together, the data indicates more intense impairment of photosynthesis in rice plants in the c ombination of the salt and heat, however the effective modulation of the antioxidative system was eff ective in establishing a new redox homeostasis and providing tolerance to abiotic stress.

Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18).

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

En la naturaleza las plantas liberan constantemente a la rizosfera una mezcla de metabolitos conocida como exudados radiculares. Su composición puede verse afectada por diferentes estímulos, incluyendo estreses abióticos como la salinidad o elevadas temperaturas. El Capítulo 1 demuestra que los portainjertos de cítricos citrange Carrizo y Citrus macrophylla exudan diferentes concentraciones de prolina y fitohormonas dependiendo del estrés abiótico y del genotipo. El Capítulo 2 estudia el efecto de dichos exudados de plantas de cítricos sometidas a salinidad y calor sobre las rizobacterias Pseudomonas putida KT2440 y Novosphingobium sp. HR1a, los cuales generalmente promueven su crecimiento. Además, se detectó la presencia de prolina y salicilatos en exudados a través del análisis de la expresión de los promotores PputA y PpahA de P. putida KT2442 y Novosphingobium sp. HR1a respectivamente. Finalmente, el Capítulo 3 muestra el efecto beneficioso de ambas bacterias en plantas de C. macrophylla sometidas a salinidad.
In nature, plants are constantly releasing a mixture of metabolites through the roots known as root exudates. Its composition can be affected by different stimuli, including abiotic stress conditions as salinity or high temperatures. Chapter 1 demonstrates that citrus rootstocks Carrizo citrange and Citrus macrophylla exude different concentrations of proline and phytohormones depending on the abiotic stress condition and the genotype. Chapter 2 studies the effect of citrus root exudates from salt- and heat-stressed plants on the rhizobacteria Pseudomonas putida KT2440 and Novosphingobium sp. HR1a, which generally promote their growth. Moreover, the presence of proline and salicylates in root exudates was also tested through the analyses of the expression of the promoters PputA and PpahA of P. putida KT2442 and Novosphingobium sp. HR1a, respectively. Finally, Chapter 3 reveals the beneficial effect of both bacterial strains in C. macrophylla plants subjected to salt stress conditions.

In this dissertation, I asked how soil biota, abiotic stress, and plant provenance influence plant communities and interactions between plants. Soil biota can have positive or negative effects on individual plants, and also influence the diversity and productivity of plant communities through their net effects on individuals and by mediating plant-plant interactions. However, the level of abiotic stress experienced by plants is likely to drive plant responses to soil mutualists and antagonists. Additionally, plant provenance (e.g. population origin) can influence responses to abiotic soil conditions as well as to soil organisms. Understanding how these three interacting components shape plant interactions may improve success of restoration and invasive plant management. During restoration, the goal is typically to create conditions conducive to native plant reestablishment. However, amelioration of disturbed areas by reducing abiotic stress or by adding beneficial soil organisms may unintentionally increase colonization and growth of non-native plants. Using the applied context of mine restoration, I examined how soil biota, abiotic stress, and plant provenance affected plant communities and interactions in four studies.

In Chapter 1, I found that both a native grass (Bouteloua gracilis ) and an invasive grass (Bromus tectorum) responded positively to soil biota when grown alone in the greenhouse. However, when grown together, the presence of soil biota increased the competitive ability of Bromus, while the removal of soil biota increased competition by Bouteloua. Results supported the hypothesis that invasive species such as Bromus often have positive responses to soil biota in the invaded range, but I also found that Bromus response to soil biota removal varied considerably by site.

In Chapters 2 and 3, I examined how methods used during restoration (application of stockpiled soil and inoculation with soil biota) affected native and non-native plant growth in field plots. I found that native plant biomass and non-native plant biomass both tended to increase when soil abiotic stress was ameliorated through the addition of deeper stockpiled soil. In addition, both native and non-native grasses responded positively to the use of local soil an as inoculant, while non-native forbs responded negatively to local soil inoculum. However, native plants only received significant benefits from inoculation when targeted application to native seedling transplants was used. Commercial mycorrhizal fungal inoculum did not affect plant growth. In studies of both stockpiled soil addition and soil inoculation, year was an important factor in determining plant responses. Variation in effects by year may reflect differences in precipitation timing or amount, or changes associated with plant and soil biota growth over time.

In Chapter 4, I used a greenhouse experiment to examine how one type of soil biota, arbuscular mycorrhizal fungi (AMF), influenced plant-plant interactions. I also manipulated abiotic stress (soil phosphorus availability) and plant provenance (stress-tolerant ecotype versus competitive ecotype) to assess whether these factors influenced AMF-mediated interactions among plants. I found that allowing or denying AMF hyphal access between neighboring pots altered plant reproduction. Inflorescence production was substantially decreased when hyphal access was allowed between two stress-tolerant plants. In addition, when hyphal access was permitted from a stress-tolerant plant to a competitive plant, the competitive plant flowered slightly sooner, whereas allowing hyphal access between two stress-tolerant plants led to slightly slower flowering. These results did not appear to be driven by abiotic stress or plant nutrition. It is possible that AMF transmission of infochemicals may play a role in regulating plant phenology and reproduction; however, further research in this area is needed.

L'Eucalyptus, feuillu le plus planté dans le monde, est fortement exposé au froid en raison de l'absence de dormance. Les gènes DREB sont connus comme étant les principaux régulateurs de la réponse aux stress abiotiques. Un nombre élevé de gènes DREB1/CBF (C-Repeat Factor) a été identifié chez Eucalyptus grandis. Le but de l'étude est de mieux comprendre le rôle de la voie DREB chez Eucalyptus pour le contrôle de la tolérance au stress, du développement et de la formation du bois. La présente étude a permis une annotation des gènes CBF et DREB2 dans le cadre d'un projet de sequençage partiel du génome d'E. gunnii. Une analyse complète de l'expression des genes par qRT-PCR a été réalisée sur les différents organes des deux espèces d'Eucalyptus après les traitements au stress. L'existence d'une copie de CBF supplémentaire dans le génome E. gunnii par rapport à E. grandis suggère que ce groupe est encore en évolution contrairement au groupe DREB2. Un nombre élevé de transcrits CBF chez E. gunnii, tolérant au froid, et forte une vitesse d'induction ce ces facteurs chez E. grandis, à croissance rapide, suggère que les facteurs CBF sont impliqués à la fois dans la protection au stress et la limitation de croissance. Des facteurs de transcription des familles MYB, NAC, KNOX et AP2/ERF impliqués dans le contrôle de la croissance et de la formation de la paroi cellulaire ont été identifiés comme étant des gènes putatifs cibles de CBF. Ces résultats sont en accord avec le phénotype modifié de surexpresseurs CBF. Les deux approches suggèrent un rôle central de la voie de DREB dans le compromis entre la croissance et la résistance au stress chez cette espèce ligneuse
Eucalyptus, the most widely planted hardwood in the world, is highly exposed to the cold due to the lack of dormancy. DREB (Drought Responsive Element Binding) genes are known as master regulators of abiotic stress response. A high number of the DREB1/CBF (C-Repeat Factor) genes has been annotated in Eucalyptus grandis. The aim of the study was to better understand the role of DREB pathway in Eucalyptus for the control of stress tolerance, development and wood formation. The present study provides an annotation of the CBF and DREB2 genes from a partial draft of the E. gunnii genome sequence. A comprehensive transcriptional analysis through high-throughput qRT-PCR was carried out on different organs from the two Eucalyptus species after stress treatments. An additional CBF copy in the E. gunnii genome compared to E. grandis suggests that this group is still evolving unlike the DREB2 group. The higher CBF transcript amounts in the cold tolerant E. gunnii together with higher induction rates in the fast growing E. grandis suggest that CBF factors promote both stress protection and growth limitation. In addition, transcription factors from MYB, NAC, KNOX and AP2/ERF families involved in the control of growth and cell wall formation have been identified as putative CBF target genes. These results are in agreement with the modified phenotype of CBF overexpressors. Both approaches suggest a central role of DREB pathway in the trade-off between growth and stress resistance in this woody species

The modification of floral characteristics will be beneficial in improving the efficiency of hybrid seed production and the breeding of more climate resilient varieties in bread wheat (Triticum aestivum). Methods for phenotyping floral traits were initially tested using small genotype panels under controlled conditions and in field trials. Low-tech phenotyping methods appropriate for use by breeders and researchers were developed and demonstrated to be accurate. A panel of 111 genotypes was subsequently assessed in field trials using these methods. A high level of genotypic variation was observed for anther extrusion, anther length and anthesis duration/pattern and phenotypes were found to be stable across trials. Using this phenotypic data set, floral trait marker-trait associations (MTAs) were detected by association mapping and additional anther extrusion quantitative trait loci (QTLs) have been detected by linkage mapping in a bi-parental population. The phenotypic effects of candidate loci co-localizing with MTAs and QTLs were investigated using TILLING mutants and modifications to floral characteristics have been observed in some mutant lines. The utilization of phenotyping and genomic resources described in the present study is discussed and areas of future research have been identified.

Mestrado em Biologia Molecular e Celular
In Portugal, Eucalyptus, particularly Eucalyptus globulus, occupies more than 800 000 ha and, due to being a major source of biomass for fiberboard, industrial charcoal, fuel wood and paper pulp, has become a key genus, with a considerable economic importance. However, E. globulus productivity faces new pressures, with climate change-driven drought as one of the most hostile ones. Drought can lead to growth impairment and yield reduction: directly; or indirectly, through the increase of plant susceptibility to pathogens by a predisposition mechanism. Neofusicoccum kwambonambiense is an endophytic opportunist phytopathogen known to severely affect E. globulus, whose incidence has already been reported in Portugal. Taking all in consideration, it is of major importance to assess the predisposition effect that drought may have on the N. kwambonambiense - E. globulus interaction. For such purpose, four treatment groups were established: E. globulus were firstly subjected to a 66-days acclimation period in which plants were periodically watered (80% of field capacity). After that, two groups were exposed to a progressive water supply restriction. The other two remained well-watered. Once water-stressed plants achieved 18% of field capacity (23 days), a well-watered and a water-stress group were inoculated with N. kwambonambiense. All treatments were kept in these conditions throughout a 65 days’ period, at which moment a set of morphological, physiological and biochemical parameters was obtained. Well-watered plants, despite being infected with N. kwambonambiense, presented an overall photosynthetic increase, which enabled plant defense through the production of sugars, proline and salicylic acid. Oxidative damages (partially observed through malondialdehyde content), were avoided in part due to proline and soluble sugars. Water stress lead to a direct growth impairment confirmed through an indole-acetic-acid content decrease. A water-potential reduction occurred, which, together with abscisic acid, lead to stomatal closure and overall photosynthetic efficiency decline. Oxidative damages weren’t properly managed and further affected E. globulus. Furthermore, N. kwambonambiense was found to promote a jasmonic acid content increase, typical of necrotrophic pathogens, which may suggest a lifestyle change from hemibiotrophic to necrotrophic as plant cells progressively degenerate. Ultimately, water-stressed E. globulus presented larger external lesion extensions and steam cankers and a superior internal fungi progression. Our results conclusively demonstrate that water stress created a better substrate for fungi development and decreased the plant’s ability to respond. Such resulted in higher susceptibility and disease severity confirming predisposition.
Em Portugal, o eucalipto, particularmente o Eucalyptus globulus, ocupa mais de 800 000 ha. Devido a ser uma importante fonte de biomassa para painéis de fibras, carvão industrial, lenha e pasta de papel, tornou-se um género chave de considerável importância económica. Contudo, a produtividade de E. globulus tem encontrado novas pressões, sendo a seca resultante das alterações climáticas, uma das mais hostis. A seca pode levar a uma diminuição do crescimento e produtividade: diretamente; ou indiretamente através do aumento da suscetibilidade a agentes patogénicos através da predisposição. O fungo ascomiceto Neofusicoccum kwambonambiense é um agente fitopatogénico endofítico oportunista que se sabe afetar severamente E. globulus, e cuja presença já fora confirmada em Portugal. Tomando tal em consideração, torna-se importante avaliar o efeito de predisposição que a seca poderá ter na interação N. kwambonambiense - E. globulus. Para tal foram criados quatro grupos de tratamento: E. globulus foram primeiramente sujeitos a um período de aclimatização de 66 dias no qual foram periodicamente irrigados (80% de capacidade de campo). Seguidamente, dois grupos foram sujeitos a uma diminuição progressiva da irrigação. Os outros dois grupos permaneceram bem regados. Uma vez que os tratamentos stressados atingiram 18% de capacidade de campo (23 dias), um grupo bem regado e um grupo stressado foram inoculados com N. kwambonambiense. Todas os tratamentos foram mantidos nestas condições durante um período de 66 dias, findo o qual foi obtido um conjunto de parâmetros morfológicos, fisiológicos e bioquímicos. As plantas bem regadas, apesar de terem sido inoculadas com N. kwambonambiense apresentaram um aumento dos parâmetros fotossintéticos o que terá permitido a defesa da planta através de uma produção amplificada de açúcares, prolina e ácido salicílico. Danos oxidativos (parcialmente observados através do conteúdo em malondialdeído) foram evitados, em parte, devido à ação da prolina e açúcares solúveis. O stress hídrico levou a uma diminuição do crescimento confirmado pela redução do conteúdo em ácido-indole-acético. Ocorreu uma diminuição do potencial hídrico, a qual, em conjunto com o aumento do ácido abscísico, levou ao fecho dos estomas e diminuição da fotossíntese. Os danos oxidativos não foram controlados, afetando o estado do E. globulus. Ademais, o N. kwambonambiense provocou um aumento do conteúdo em ácido jasmónico, típico de agentes patogénicos necrotróficos, o que poderá sugerir que o fungo passou de um estilo de vida hemibiotrófico para necrotrófico, à medida que as células degeneravam. Os E. globulus stressados apresentavam maiores lesões externas e cancros, conjuntamente com uma maior progressão interna do fungo. Os nossos resultados comprovam que a seca criou um melhor substrato para o desenvolvimento do fungo e diminuiu a capacidade de resposta da planta. Tal resultou num aumento da suscetibilidade e severidade da doença confirmando a predisposição.

Abiotic stresses are one of the major limiting factors of plant growth and thus crop productivity. Exposure to these stresses, including temperature and UV, cause physiological and epigenetic changes in plants. Such changes may be inherited in the progeny of stressed plants, and may change their ability to respond to stress. To understand the ability of plants to inherit an epigenetic stress memory as well as the physiological manifestations of such a memory, we propagated both stressed and control plants and compared the progeny under both normal and stressed conditions. In addition to wild-type plants we used Dicer-like mutants dcl2, dcl3 and dcl4, as Dicers have been linked to RNA-directed DNA methylation, a form of epigenetic memory. These studies revealed that leaf number decreases in the progeny of stressed plants, and bolting occurs earlier in the progeny of temperature stressed plants but later in the progeny of UV-C stressed plants. Transposons were also re-activated in the progeny of stressed plants. While heat shock transcription factor 2A increased expression in the progeny of heat stressed plants, many genes involved in DNA repair and histone modifications decreased. DCL2 and DCL3 appeared to be more important in transgenerational stress memory than DCL4. However, all dcl plants were generally not significantly different than wild-type plants, indicating that a single DCL deficiency may be compensated for by another DCL.
xiv, 246 leaves : ill. ; 29 cm

Doctor of Philosophy
Department of Horticulture, Forestry, and Recreation Resources
Sunghun Park
Plants are continuously exposed to numerous abiotic stresses, which adversely affect plant growth, development, and yield. Plants have developed different signaling pathways to cope with abiotic stresses, and some of the pathways converge to help plants tolerate simultaneous stresses. Here, we report ectopic expression of an Arabidopsis glutaredoxin AtGRXS17 that confers tolerance to multiple abiotic stresses in tomato plants. In yeast assays, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double mutants to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. Furthermore, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing tomato plants during heat stress when compared to wild-type controls. Under cold, drought, and oxidative stress conditions, AtGRXS17-expressing tomato plants also displayed more vigorous growth and less physiological damage than those of the wild-type control plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that expression of AtGRXS17 alters multiple stress defense signaling pathways, including the Abscisic Acid (ABA) and C-Repeat Binding Factors (CBF) pathways. The results revealed a conserved function for a glutaredoxin protein in abiotic stress adaptation, and manipulation of AtGRXS17 may be a useful approach to improve crop stress tolerance and understand plant signaling under abiotic stress conditions. Deregulated expression of an Arabidopsis H[superscript]+/Ca[superscript]2[superscript]+ antiporter (sCAX1) in agricultural crops increases total calcium (Ca[superscript]2[superscript]+) but may result in yield loses due to calcium-deficiency like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca[superscript]2[superscript]+ binding protein located at endoplasmic reticulum) in sCAX1-expressing plants mitigated these adverse effects while maintaining enhanced Ca[superscript]2[superscript]+ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca[superscript]2[superscript]+ transporters and binding protein at different intracellular compartments can alter the content and distribution of calcium within the plant matrix.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites.
AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

Increases in temperature damage plant cells, and plants react to heat stress by inducing of a number of protective mechanisms. In this study, it has been shown that heat damages cells both directly while the plants are being heated, and indirectly through heat induced oxidative stress during recovery from heating. Different stress response pathways are induced in each case. Evidence implicates salicylic acid, abscisic acid, ACC (a precursor of ethylene), calcium ions and active oxygen species in pathways resulting in thermotolerance (i.e. increased survival at high temperature). Addition of these potential second messengers results in increased survival and decreased oxidative damage after heating (as measured using the TBARS assay), while silencing the pathways through use of mutants, inhibitors or transgenes results in decreased thermotolerance. In vivo calcium measurements show cytosolic calcium transients only at initiation of cooling after heat stress. Calcium chloride also induces thermotolerance when added after heating, as does ACC. These substances can induce early increases in ascorbate peroxidase activity after heating, and induce expression of antioxidant genes. Thus they may play a role in heat induced oxidative stress response pathways in recovery. Two different pathways induced during heating appear to result in the expression of genes for heat shock proteins. The predominant pathway induced at 30oC involves ABA, while that at 35-40oC involves an oxidative burst generated through the NADPH oxidase, atrbohB. Addition of SA can induce HSP (heat shock protein) expression, but there is little evidence that this occurs endogenously in plant cells, although nahG plants unable to signal via SA are thermosensitive. Results in this study indicate that there are several signalling pathways associated with heat shock. The pathway induced during recovery from heating involves calcium ions and ethylene, and results in increased antioxidant capacity. During heating two pathways induce expression of HSPs: one involving abscisic acid and possibly salicylic acid and one involving an oxidative burst. At least one further pathway is believed to exist, which involves protein kinases and phosphatases as heat shock causes up-regulation of expression of certain genes for these signalling components.

ABSTRACT Bacteriocins are small peptides, produced by many bacteria, which have adverse effects on microorganisms closely related to the producer strain. Two new Class IId bacteriocins, thuricin 17 and bacthuriocin F4 were isolated from plant growth promoting rhizobia (PGPR). Initial studies showed that both the bacteriocins can promote soybean growth. My experimental results demonstrated that these two bacteriocins promoted germination and emergence of corn, soybean, canola and wheat, but had no effect on barley, when compared with the control under optimal growth conditions. Of the tested crops, corn was most positively affected. Further abiotic stress experiments on corn showed that the two bacteriocins promoted corn emergence under low temperature, high salinity and polyethylene glycol induced water deficit stress. Field experiment data also showed that thuricin 17 can effectively promote corn emergence under cool spring field conditions in southwestern Quebec.
RÉSUMÉ Les bacteriocines sont les petits peptides produits par les bactéries qui ont des effets défavorables sur les microorganismes génétiquement proches de la souche productrice. Deux nouvelles bacteriocines de la classe IId, thuricin 17 et bacthuriocin F4, ont été isolées de bactéries présentes dans la rhizosphere et qui promouvoit la croissance des plantes (PGPR). Des études préalables ont démontré que ces deux bacteriocines peuvent promouvoir la croissance du soja. Mes résultats expérimentaux ont démontré que ces deux bacteriocines promouvoit la germination et l'émergence du maïs, du soja, du canola et du blé, mais n'ont pas d'effet sur l'orge, lorsque comparées aux contrôles sous des conditions optimales pour la croissance. Parmi les cultures testées, le maïs était le plus positivement affecté. Les expériences subséquentes sous stress abiotiques ont démontré que les deux bacteriocines ont augmenté l'émergence du maïs sous conditions de basse température, de salinité et de stress hydrique provoqué par le glycol de polyéthylène. Aussi, les expérimentations en champ ont démontré que le thuricin 17 peut promouvoir efficacement l'émergence du maïs dans le sud-ouest du Québec, où les températures printanières sont souvent fraîches.

Invasive species are cited as a major threat to native community composition and biodiversity throughout the world. Some recent studies have focused on whether invasive species are the drivers or passengers of change in degraded systems. A strongly-interactive community is supposed to resist invasion by all but the most strong invasive competitors (`drivers') which can establish there without the aid of disturbance and actively reduce the abundance of natives. A weakly-interactive community, impaired by some anthropogenic disruption, is invasible by weaker exotics which are merely `passengers' of the habitat degradation that is more constraining to natives. Though competitive and disturbance-adapted species fit into this model, there is no correlate for species with superior toleration of stress. Systems with high degrees of natural abiotic stress are weakly-interactive and as a result may be invasible by an exotic `tolerator' in the same way that anthropogenically-weak systems are invasible by passengers. Dolomite prairie, differentiated from typic tallgrass prairie by its shallow soils, represents a relatively stressful system. A study of its plant community composition and relationships to environmental variables was done to get a better understanding of the natural abiotic drivers of composition. Compositional patterns were most closely associated with the soil depth gradient. Exotic Poa species were by far the most frequent invaders, a finding more typical of Great Lakes alvars than of typic tallgrass prairie of which the dolomite prairie is a subset. I hypothesized that Poa species dominated dolomite prairie via the tolerator model. In a series of removal treatments, I determined that Poa is neither a driver nor a tolerator, but a passenger of environmental degradation. My results suggest that historic anthropogenic degradation rather than specific competitive ability is a common explanation for exotic dominance on the landscape today. More empirical work needs to be conducted in other stressful (particularly relatively undisturbed) systems to further investigate the tolerator model.

Drought is one of the major causes of food insecurity across the globe, particularly in India and the Asia Pacific, where subsistence agriculture is a major means of survival. Pulses such as mungbean and chickpea are staple foods in these regions and their production is especially affected by drought. This study targeted plant roots as a way of enhancing drought tolerance using a newly developed chemical treatment. Some strategies included advanced plant root imaging, agricultural modelling, bioinformatics and large scale field trials. This may allow stabilization of crop production which is increasingly coming under threat by drought and climate change.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
O início da senescência foliar é uma mudança do desenvolvimento altamente regulada, que é controlada por ambos genética e ambiente. A senescência é desencadeada por reprogramação massiva de transcrição, mas informações funcionais sobre os seus mecanismos reguladores subjacentes é limitada. No presente estudo, foi realizada uma análise funcional do fator de transcrição de soja (Glycine max) GmNAC081 induzido por estresse osmótico e do retículo endoplasmático (ER), durante senescência foliar natural, utilizando estudos de superexpressão e genética reversa. Linhagens superexpressando GmNAC081- exibiram floração e senescência foliar aceleradas, mas de outra forma se desenvolveram normalmente. A senescência foliar precoce de linhagens superexpressando GmNAC081 foi associada com maior perda de clorofila, deterioração fotossintética mais rápida e maior expressão de genes de codificação de enzimas hidrolíticas alvo de GmNAC081, incluindo a enzima de processamento vacuolar (VPE), uma executora da morte celular programada (PCD) desencadeada no vacúolo. Por outro lado, o silenciamento de GmNAC081 mediado por VIGS adia a senescência foliar e foi associado com reduções na perda de clorofila, a peroxidação lipídica e a expressão de alvos diretos de GmNAC081. Os estudos do promotor-repórter revelaram que o padrão de expressão GmNAC081 foi associado com a senescência, em folhas de soja. Nossos dados indicam que GmNAC081 é um regulador positivo da senescência dependente da idade e pode integrar as respostas de MCP induzida por estresse osmótico, do RE e senescência natural, completando o circuito regulamentar o GmNAC081 / VPE. Além de estressores ambientais, muitas espécies de plantas são sensíveis a concentrações micromolares de Al. Em sorgo, o gene de resistência ao alumínio é SbMATE, altamente expresso no ápice da radícula e codifica um transportador de membrana que pertencem à família MATE (multidrug and toxic compound extrusion family), que é responsável pelo efluxo citrato ativado por alumínio. Nesta espécie a região codificadora do gene de tolerância ao alumínio é identica entre cultivares tolerantes e sensíveis. Além de um polimorfismo encontrado no segundo íntron, elementos transponíveis do tipo MITE foram detectados na região promotora, sendo o número de repetições MITE positivamente relacionada com a tolerância. Neste trabalho, a análise in silico do promotor de SbMATE apresentou sequencias típicas de promotores eucarióticos assim como a presença de elementos cis regulatórios que conferem tolerância ao aluiminio em Arabidopsis. A ausência de atividade de promotores inteiros transformados em Arabidopsis sugerem a presença de elementos cis regulatórios negativos reprimindo a atividade em Arabidopsis ou ainda, a presença de silenciamento transcricional por RNA de interferência em Arabidopsis que não ocorre em Sorghum. Deleções do promotor, mostraram que os repressores estão localizados na sequência do elemento transponível já que na ausência deste elemento, o promotor passa a ser ativo em todas as partes da planta de forma independente da idade. A prospecção por fatores de transcrição que controlam a capacidade de resposta do gene SbMATE de Sorghum e a análise da interação entre prováveis transfatores com o promotor de SbMATE, foram realizadas através do sistema mono-hibrido em leveduras e evidenciaram a necessidade da obtenção de uma nova biblioteca de cDNA confeccionada por um kit mais adequado à esta planta, além da utilização de diferentes vetores de clonagem para as análises das iterações.
The onset of leaf senescence is a highly regulated developmental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcriptional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC081 during natural leaf senescence using overexpression studies and reverse genetics. GmNAC081- overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC081-overexpressing lines was associated with greater chlorophyll loss, faster photosynthetic decay and higher expression of hydrolytic enzyme- encoding GmNAC081 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered programmed cell death (PCD). Conversely, VIGS-mediated silencing of GmNAC081 delayed leaf senescence and was associated with reductions in chlorophyll loss, lipid peroxidation and the expression of GmNAC081 direct targets. Promoter–reporter studies revealed that the expression pattern of GmNAC081 was associated with senescence in soybean leaves. Our data indicate that GmNAC081 is a positive regulator of age-dependent senescence and may integrate osmotic stress- and ER stress-induced PCD responses with natural leaf senescence thorough the GmNAC081/VPE regulatory circuit. In addition to environmental stressors, many plant species are sensitive to micromolar concentrations of Al. In sorghum, aluminum resistance SbMATE gene is highly expressed at the apex of the radicle and encodes a membrane transporter belonging to MATE family (multidrug and toxic Compound Extrusion family), which is responsible for the efflux activated aluminum citrate. In this species the coding region of the aluminum tolerance gene is identical between tolerant and sensitive cultivars. In a polymorphism is found in the second intron, the MITE type transposable elements were detected in the promoter region, the number of repetitions MITE positively related tolerance. In this work, the analysis in silico showed typical SbMATE promoter sequences of eukaryotic promoters, as well as the presence of cis regulatory elements that confer tolerance in Arabidopsis. The absence of whole promoter activity in transformed Arabidopsis suggesting the presence of negative regulatory cis- elements repressing the activity in Arabidopsis or the presence of transcriptional silencing by RNA interference in Arabidopsis does not occur in sorghum. Deletions promoter showed that the repressors are located in sequence as the transposable element in the absence of this element, the promoter becomes active in every part regardless of the age of plant form. Prospecting for transcription factors that control the responsiveness of SbMATE Sorghum gene and analysis of the interaction between likely transfatores with SbMATE promoter, were performed using mono-hybrid system in yeast and demonstrated the need for obtaining a new cDNA library made for a more appropriate for this plant kit, besides the use of different cloning vectors for analysis of iterations.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010.
ABSTRACT: Nitric Oxide (NO) is an essential bioregulatory molecule in plant growth, development, and tolerance against biotic and abiotic stresses. In legume root nodules, abiotic stresses impose restraint on metabolic capacity of bacteria and cause oxidative damage to cellular macromolecules, leading to inhibition of nitrogenase activity. In this study, the primary aim was to determine the influence of NO signaling on cysteine protease activity in soybean (Glycine. max [L] Merr) root nodules. Intact plants were treated with a NO donor, diethylenetriamine/nitric oxide adjunct (DETA/NO), 8-(4-chlorophenylthio)-[CPT]- cGMP, sorbitol and sodium chloride (NaCl). The root nodule cysteine protease activity was measured using the chromogenic substrate N-benzoil-L-p-nitroanilide (L-BAPNA). The results demonstrated NO as acting both as a protection against programmed cell death (PCD) at low exogenously applied NO concentrations, or as inducing PCD through regulating the cysteine proteases activity in root nodules when NO is applied at elevated concentrations. In the root nodules, the activity of cysteine protease is regulated either through cyclic guanosine monophosphate (cGMP)-dependent during abiotic stress or cGMP-independent pathways during normal root nodule development. The purpose of this research was to highlight the importance of NO in cell signaling and cysteine protease activity in legume root nodules. We also focused on the effect of abiotic stress on two maize genotypes as well as the influence of abiotic stress on cysteine protease activity in the abiotic stress-sensitive maize genotype than the tolerant genotype. The study suggests that cysteine protease activity can be used as early screen to identify abiotic stress-sensitive/tolerant maize genotype upon exposure to abiotic stress.
AFRIKAANSE OPSOMMING: Geen Afrikaanse opsomming beskikbaar.
National Research Foundation

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll content show no significant difference between the wild-type plants and sip428-silenced transgenic plants. In summary, based on the results presented in this study it could be concluded that SIP428 is a negative regulator of salinity, osmotic and drought stresses. Further studies are required to understand the mechanism.

In the first part of the work the behaviour of different light harvesting complex (Lhc) subunits, belonging to a highly conserved multigenic family, was analysed in response to different growth conditions in Zea mays. The redundancy of these sequences suggested, in fact, a possible specific role of each gene product in light harvesting and photoprotection, depending on environmental conditions. Plants were grown in different conditions of light and temperature and thylakoid membranes were isolated in order to test the accumulation of different Lhc proteins. Significant differences were found in the accumulation of both major (LHCII) and minor antennae of Photosystem II (PSII). Temperature seemed to play an important role, since the LHCII/minor antenna ratio increased with decreasing temperature, suggesting that the rate of light energy utilization vs excess energy was the driving force for these changes. Low temperature, in fact, is a powerful stress factor since it greatly decreases the electron transport rate at the step of QH2 oxidation by Cyt b6f thus leading to over-reduction of the PQ pool; moreover the decrease of Calvin cycle activity depresses ATP and NADPH utilisation thus making NADP+ unavailable as electron acceptor from PSI. The effect of low temperature is particularly stressing in the case of C4 plants because of their tropical origin. The observed changes included significant variations in pigment composition and activity of Non Photochemical Quenching. These results confirmed the specific role of different antennae in the organization of the Photosystem II and photoprotection. In the second part of this thesis, the attention was focused on the role in acclimation of two major components of stress: ROS production and plastoquinone reduction. To elucidate this problem, the modulation of antenna polypeptides following environmental conditions was analysed using a barley (Hordeum vulgare) mutant, viridis zb63, which lacks Photosystem I, to mimic extreme and chronic over-excitation of Photosystem II. [. . . ]
Dans la première partie du travail, il a été analysé le comportement des différents subunités appartenantes à la famille multigénique du Lhcb, dans réponse à des différentes conditions de croissance en maïs (Zea mays). La redondance de ces séquences, en effet, a suggéré un possible rôle spécifique de chaque produit génique dans la capture de la lumière et dans la photoprotection, sur la base des conditions environnementales. Les plantes ont été poussées en différentes conditions de lumière et température, pour ensuite en isoler les tylacoides au fin de les tester pour l’accumulation des protéines des antennes (Lhc). Des significatives différences ont été relevées dans les antennes, soit majeurs (LHCII) que mineurs, du Photosystème II (PSII) et, en détail, la température a joué un important rôle, puisque le rapport LHCII/antennes mineures a montré une augmentation avec la diminution de la température. Le froid, en effet, est un facteur de stress assez puissant, dès qu’il réduit énormément la vitesse de transport des électrons au niveau de l’oxydation du QH2 de la part du Cytb6f, en déterminant un excès de réduction du plastoquinone. Dans la deuxième partie de cette thèse, la régulation par les conditions environnementales des polypeptides qui constituent l'antenne photosynthétique a été analysée en employant un mutant d’orge (Hordeum vulgare), viridis zb63, qui manque du Photosystème I, ce qui produit la réduction du plastoquinone et mime une surexcitation chronique du Photosystème II. [. . . ]

FARIAS, Gabriel Castro. Biometria e respostas fisiológicas de feijão-de-corda inoculado com fungos endofíticos dark septate sob estresse salino. 2014. 55 f. Dissertação (Mestrado em engenharia agrícola)- Universidade Federal do Ceará, Fortaleza-CE, 2014.
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With the increasing of areas degraded by soil salinization process, is necessary to increase of agricultural investments to be able to reach a production acceptable. In order to reduce such inputs, studies of microorganisms involved in the production process are becoming more constant. The objective of this study was to evaluate the associations between endophytic fungi Dark Septate and cowpea plants when irrigated with water of different salinities, analyzing biometrics, gas exchange, nutrient content and accumulation of organic solutes in the leaves. Cowpea plants were grow in containers of five liters under greenhouse conditions and subjected to four different electrical conductivity of irrigations water (1.2; 2.2; 3.6; 5.0 dS m-1). Were used for the inoculation strains B’2 and 21W and treatments were as follows: F1 – plants that were not inoculated; F2 – plants inoculated with strains 21W and F3 – plants inoculated with strain B’2. during the experiment was measured gas exchange and at the end of the experiment the plant were collected and determinate the leaf area, shoot dry mass, root dry mass, concentration of the foliar nutrients and organic solutes, and was made the determination colonization of roots by the fungus. The salinity caused significant reductions in biomass, leaf area and leaf gas exchange, however, the presence of the endophytic fungi provided a large tolerance to the adverse effects of salinity. There was a larger accumulation of calcium, sodium, clore and phosphorus in cowpea leaves with increasing salinity and decrease in the leaves of nitrogen and potassium. The presence of the fungus in the plant provided a foliar phosphorus and nitrogen.
Com o crescimento das áreas degradadas pelo processo de salinização dos solos se faz necessário o aumento do uso de insumos para que seja possível atingir uma produção aceitável. Visando a redução de tais insumos, estudos sobre microrganismos envolvidos no processo produtivo são cada vez mais constantes. O objetivo deste trabalho foi avaliar a resposta das associações entre fungos endofíticos Dark Septate e plantas de feijão-de-corda quando irrigado com águas de diferentes salinidades, analisando-se a biometria, troca gasosas, teores foliares de nutrientes e o acúmulo de solutos orgânicos nas folhas. As plantas de feijãode-corda foram cultivadas em vasos de 5 litros, sob condições de casa de vegetação e submetidas a quatro diferentes condutividades elétricas da água de irrigação (1,2; 2,2; 3,6 e 5,0 dS m-1). Para a inoculação foram utilizados as linhagens B’2 e 21W e os tratamentos foram os seguintes: F1 – plantas que não foram inoculadas; F2 – plantas inoculadas com a linhagem 21W e F3 – plantas inoculadas com a linhagem B'2. Durante a condução do experimento mediu-se as trocas gasosas e ao final do experimento as plantas foram coletadas e determinou-se a sua área foliar, massa seca da parte aérea, massa seca das raízes teores de nutrientes e solutos orgânicos foliares, bem como foi feita a determinação de colonização nas raízes pelo fungo. A salinidade provocou reduções significativas na biomassa, área foliar e trocas gasosas, contudo, a presença do fungo endofítico proporcionou uma maior tolerância aos efeitos negativos da salinidade. Houve um maior acúmulo de cálcio, sódio, cloro e fósforo em folhas de feijão-de-corda com o aumento da salinidade e uma diminuição dos teores de nitrogênio e potássio. A presença do fungo na planta proporcionou maior teor foliar de fósforo e nitrogênio.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
With the increasing of areas degraded by soil salinization process, is necessary to increase of agricultural investments to be able to reach a production acceptable. In order to reduce such inputs, studies of microorganisms involved in the production process are becoming more constant. The objective of this study was to evaluate the associations between endophytic fungi Dark Septate and cowpea plants when irrigated with water of different salinities, analyzing biometrics, gas exchange, nutrient content and accumulation of organic solutes in the leaves. Cowpea plants were grow in containers of five liters under greenhouse conditions and subjected to four different electrical conductivity of irrigations water (1.2; 2.2; 3.6; 5.0 dS m-1). Were used for the inoculation strains Bâ2 and 21W and treatments were as follows: F1 â plants that were not inoculated; F2 â plants inoculated with strains 21W and F3 â plants inoculated with strain Bâ2. during the experiment was measured gas exchange and at the end of the experiment the plant were collected and determinate the leaf area, shoot dry mass, root dry mass, concentration of the foliar nutrients and organic solutes, and was made the determination colonization of roots by the fungus. The salinity caused significant reductions in biomass, leaf area and leaf gas exchange, however, the presence of the endophytic fungi provided a large tolerance to the adverse effects of salinity. There was a larger accumulation of calcium, sodium, clore and phosphorus in cowpea leaves with increasing salinity and decrease in the leaves of nitrogen and potassium. The presence of the fungus in the plant provided a foliar phosphorus and nitrogen.
With the increasing of areas degraded by soil salinization process, is necessary to increase of agricultural investments to be able to reach a production acceptable. In order to reduce such inputs, studies of microorganisms involved in the production process are becoming more constant. The objective of this study was to evaluate the associations between endophytic fungi Dark Septate and cowpea plants when irrigated with water of different salinities, analyzing biometrics, gas exchange, nutrient content and accumulation of organic solutes in the leaves. Cowpea plants were grow in containers of five liters under greenhouse conditions and subjected to four different electrical conductivity of irrigations water (1.2; 2.2; 3.6; 5.0 dS m-1). Were used for the inoculation strains Bâ2 and 21W and treatments were as follows: F1 â plants that were not inoculated; F2 â plants inoculated with strains 21W and F3 â plants inoculated with strain Bâ2. during the experiment was measured gas exchange and at the end of the experiment the plant were collected and determinate the leaf area, shoot dry mass, root dry mass, concentration of the foliar nutrients and organic solutes, and was made the determination colonization of roots by the fungus. The salinity caused significant reductions in biomass, leaf area and leaf gas exchange, however, the presence of the endophytic fungi provided a large tolerance to the adverse effects of salinity. There was a larger accumulation of calcium, sodium, clore and phosphorus in cowpea leaves with increasing salinity and decrease in the leaves of nitrogen and potassium. The presence of the fungus in the plant provided a foliar phosphorus and nitrogen.

Xenobiotics are toxic chemicals that are normally not the natural substrates for enzymes or transporters involved in plant resistance. Plants have developed a three phases detoxification system from toxic compounds. Xenobiotic are firstly activated so that certain functional groups can be exposed to the successive action of several modifying enzymes. Among them, the glutathione transferases (GSTs) catalyze the nucleophilic addition of glutathione (GSH) to the electrophilic groups of a large variety of hydrophobic toxic molecules. Previously, two gstu genes have been isolated from sweet orange leaves [(Citrus sinensis) L. Osbeck)] namely GSTU1 and GSTU2. The encoded proteins differ in three amino acids, all of them included in the C-terminal domain of the enzymes (R89P, E117K, I172V). In order to evaluate the contribution of the mismatched amino acids on the catalytic activity of enzymes, several cross-mutant genes were produced by site-directed mutagenesis followed by the biochemical characterization of the in vitro expressed enzymes. In this work, transgenic tobacco plants via Agrobacterium tumefaciens mediated transformation over-expressing both the wild type and mutant CsGSTU genes were generated. Along with the molecular characterization of transformed plants, an in planta study to assess their ability in detoxifying herbicides was also performed. Therefore, transgenic plants were subjected to the action of fluorodifen, a diphenyl ether herbicide that cause photooxidative stress by inhibition of the plastid protoporphyrinogen oxidase and alachlor a chloroacetanilide herbicide which is used to control the growth of broad-leafed weeds and grasses in many crops. The electrolytic leakage assay was carried out to test the damage caused by fluorodifen treatment upon transformed and untransformed tobacco plants. The data revealed that the transgenic lines show a sharp reduction of membrane damage compared with the wild type tobacco plants. To study the tolerance towards alachlor in planta, we assayed the growth inhibition of untrasformed wild type and transgenic tobacco seedlings in the presence of 7.5 mg/l of alachlor. Alachlor negatively influences the growth of roots and stems of untransformed an transformed tobacco seedlings with the exception of the transgenic plants over-expressing CsGSTU2 which are clearly unaffected by herbicide treatment considering either stem or root lenght. Consequently, the herbicide-tolerant transgenic tobacco plants, which are described in the present study, can be utilized for phytoremediation of residual xenobiotics in the environment. Drought and salinity stress tolerance was also assessed. When exposed to 200 mM NaCl both the wild type and transgenic seedlings exhibit a reduction of root lenght, with the exception of the CsGSTU2 over-expressing tobacco line whose root length is as long as untreated control roots indicating a high level of tolerance to NaCl. The effect of drought stress upon root elongation was measured by growing seedlings in the presence of 8% mannitol. In this case all treated tobacco seedlings disclose a sharp decrease of root length, although transgenic lines appear to better tolerate drought stress conditions as the mean root length is significantly higher than that of treated tobacco wild type seedlings. In order to understand the response of tobacco plants over-expressing the CsGSTU genes to biotic stress, untransformed and transformed tobacco leaves were infiltrated with a bacterial suspension of the P. syringae pv. tabaci Tox+ DAPPG-PG 676 strain. The differences observed in symptomatology indicate that the over-expression of CsGSTU1 and CsGSTU2 in tobacco plant bestow the capability to avoid active toxin diffusion in plant tissues blocking chlorotic halos formation probably because tabtoxin is head towards a modification pathway in which CsGSTs could be involved in. This result was confirmed when tobacco leaves was treated with culture filtrates.