Academic literature on the topic 'Plant abiotic stresses'

Extreme weather events are one of the biggest dangers posed by climate breakdown. As the temperatures increase, droughts and desertification will render whole regions inhospitable to agriculture. At the same time, other regions might suffer significant crop losses due to floods. Usually, regional food shortages can be covered by surpluses from elsewhere on the planet. However, the climate breakdown could trigger sustained food supply disruptions globally. Therefore, it is necessary to develop more stress-resilient crop alternatives by both breeding new varieties and promoting underutilized crop species (orphan crops). The articles in this special issue cover responses of staple crops and orphan crops to abiotic stresses relevant under the climate breakdown, such as heat, water, high salinity, nitrogen, and heavy metal stresses. This information will certainly complement a toolkit that can help inform, support, and influence the design of measures to deal with the climate crisis.

In many parts of the world, the agricultural sector is faced with a number of challenges including those arising from abiotic environmental stresses which are the key factors responsible for most reductions in agrifood production. Crude oil contamination, an abiotic stress factor and a common environmental contaminant, at toxic levels has negative impacts on plants. Although various attempts have been made to demonstrate the impact of abiotic stresses on crops, the underlying factors responsible for the effects of crude oil and its induced abiotic stresses on the composition of the stressed plants are poorly understood. Hence, this review provides an in-depth examination of the: (1) effect of petroleum hydrocarbons on plants; (2) impact of abiotic environmental stresses on crop quality; (3) mechanistic link between crude oil stress and its induced abiotic stresses; as well as (4) mode of action/plant response mechanism to these induced stresses. The paper clearly reveals the implications of crude oil-induced abiotic stresses arising from the soil-root-plant route and from direct application on plant leaves.

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil microorganisms that can stimulate plant growth and increase tolerance to biotic and abiotic stresses. Some PGPR are capable of secreting exopolysaccharides (EPS) to protect themselves and, consequently, their plant hosts against environmental fluctuations and other abiotic stresses such as drought, salinity, or heavy metal pollution. This review focuses on the enhancement of plant abiotic stress tolerance by bacterial EPS. We provide a comprehensive summary of the mechanisms through EPS to alleviate plant abiotic stress tolerance, including salinity, drought, temperature, and heavy metal toxicity. Finally, we discuss how these abiotic stresses may affect bacterial EPS production and its role during plant-microbe interactions.

Brassicaceae plants, as an important source of primary and secondary metabolites, are becoming a research model in plant science. Plants have developed different ways to ward off environmental stress factors. This is lead to the activation of various defense mechanisms resulting in a qualitative and/or quantitative change in plant metabolite production. Reactive oxygen species (ROS) is being continuously produced in cell during normal cellular processes. Under stress conditions, there are excessive production of ROS causing progressive oxidative damage and ultimately cell death. Despite their destructive activity, ROS are considered as important secondary messengers of signaling pathway that control metabolic fluxes and a variety of cellular processes. Plant response to environmental stress depends on the delicate equilibrium between ROS production, and their scavenging. This balance of ROS level is required for performing its dual role of acting as a defensive molecule in signaling pathway or a destructive molecule. Efficient scavenging of ROS produced during various environmental stresses requires the action of several non-enzymatic as well as enzymatic antioxidants present in the tissues. In this review, we describe the ROS production and its turnover and the role of ROS as messenger molecules as well as inducers of oxidative damage in Brassicaceae plants. Further, the antioxidant defense mechanisms comprising of enzymatic and non-enzymatic antioxidants have been discussed. Keywords: Abiotic stress, Antioxidant defence, Brassicaceae, Oxidative stress, ROS

Rice as a sensitive crop that usually affected by many harmful environmental stresses. Numerous policies are followed to increase plant growth-tolerance under abiotic-stresses in various plant species. The attempts to improve crop tolerance against abiotic stresses via common breeding method are needed to follow a long-term, and may also be non-affordable, these are due to the existing genetic variability of the plant. Current review analysis existing knowledge gaps, challenges, and opportunities in the biochar application as a beneficial and pyrogenic-C, material. Consequently, a review of the literature with a high focusing on the multiple beneficial effects of using biochar on tolerance and productivity of rice in abiotic stresses is needed. This review provides a summary of those efforts that would be beneficial in reducing inconvenienced abiotic-stresses, and also how using biochar could increase rice tolerance and production through the supporting of plant growth regulator's roles. Accordantly, present review findings showed that biochar is a great amendment and consisting of principally organic rich-C matter, which has multiple benefits on improving soil physicochemical and biological properties as well as increasing rice tolerance and its productivity through enhancing plant hormones roles under abiotic stressed conditions (heat/cold temperature, drought, salinity, heavy metal, and climate change stresses). Nevertheless, it is anticipated that further researches on the benefits of biochar will increase the comprehension of interactions between biochar and plant growth hormones, to accelerate our attempts for improving rice tolerance and productivity, under abiotic-stress conditions.

Plants are exposed to various environmental stresses that affect crop growth and production. During stress, various physiological and biochemical changes including the production of nitric oxide (NO), take place. It is clear that NO could work through either transcriptional or post-translational level. The redox-based post-translational modification S-nitrosylation – the covalent attachment of an NO moiety to a reactive cysteine thiol of a protein to form an S-nitrosothiol (SNO) – has attracted increasing attention in the regulation of abiotic stress signalling. So far, the relevance of S-nitrosylation of certain proteins has been investigated under abiotic stress. In this work, we focus on the current state of knowledge regarding S-nitrosylation in plants under abiotic stress, and provide a better understanding of the relevance of S-nitrosylation in plant response to abiotic stress.

Climate change significantly affects plant growth and productivity by causing different biotic and abiotic stresses to plants. Among the different abiotic stresses, at the top of the list are salinity, drought, temperature extremes, heavy metals and nutrient imbalances, which contribute to large yield losses of crops in various parts of the world, thereby leading to food insecurity issues. In the quest to improve plants’ abiotic stress tolerance, many promising techniques are being investigated. These include the use of nanoparticles, which have been shown to have a positive effect on plant performance under stress conditions. Nanoparticles can be used to deliver nutrients to plants, overcome plant diseases and pathogens, and sense and monitor trace elements that are present in soil by absorbing their signals. A better understanding of the mechanisms of nanoparticles that assist plants to cope with abiotic stresses will help towards the development of more long-term strategies against these stresses. However, the intensity of the challenge also warrants more immediate approaches to mitigate these stresses and enhance crop production in the short term. Therefore, this review provides an update of the responses (physiological, biochemical and molecular) of plants affected by nanoparticles under abiotic stress, and potentially effective strategies to enhance production. Taking into consideration all aspects, this review is intended to help researchers from different fields, such as plant science and nanoscience, to better understand possible innovative approaches to deal with abiotic stresses in agriculture.

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.

AbstractAbiotic stresses, such as drought and high temperature, significantly limit wheat yield globally and the intensity and frequency of these stresses are projected to increase in most wheat growing areas. Wheat breeders have incrementally improved the tolerance of cultivars to these stresses through empirical selection in the environment, however new phenotyping and genetic technologies and strategies can significantly improve rates of genetic gain. The integration of new tools and knowledge in the plant breeding process, including better breeding targets, improved choice of genetic diversity, more efficient phenotyping methods and strategy and optimized integration of genetic technologies in the context of several commonly used wheat breeding strategies is discussed. New knowledge and tools that improve the efficiency and speed of wheat improvement can be integrated within the scaffold of most wheat breeding strategies without significant increase in cost.

Laboratory and field experiments evidence that silicon fertilizers contribute to plant tolerance to unfavorable growth conditions: drought, frost, salinity, heavy metal contamination, and others. Silicon-induced underlying mechanisms include thickening of the epidermal layer, enhanced root system development, chemical stability of the DNA, RNA, and chlorophyll molecules, improved transport and redistribution of elements, as well as activation of defense system in plants against oxidative damage. Application of Si fertilizers and biostimulators promoted reducing crop losses and increasing yield of rice, wheat, barley, soya, potatoes and others under drought and frost conditions.

Breeding is aimed to breed for varieties that are tolerant against disease, suitable for mechanized cultivation and harvest, and also offer them to the farmers as promising varieties. Since the purpose of legumes production is to obtain grain products of high yield and quality, developing suitable varieties for target regions where they will be grown is an important factor that needs to be considered. This breeding program aimed to develop new variety of recommended legumes varieties for different regions and will stimulate an increase in cultivation area. In Turkey chickpea is traditionally sown in spring and subjected to drought and heat stresses. Chickpea can be sown in autumn with new cultivars but winter-sown chickpea cultivars are not available for highlands. Some abiotic stresses (drought, heat, freezing etc.) and some biotic stresses (ascochyta blight, Fusarium wilt, and weeds) are common and important stresses, whereas nutrient imbalance includ-ing salinity are localized challenges. Lentil is usually traditionally sown in autumn and Central Anatolia green lentil, South Eastern Anatolia red lentil regions in Turkey. As a result of Turkish food legume Program, 48 chickpea, 41lentil, 49 beans, 3 faba beans, 3 pea and 4 cowpea varieties were registered.

Wheat (Triticum aestivum L.) is one of the most important cereal food crops worldwide. Various abiotic and biotic stresses or their combinations lead to crop losses (up to 50-82%) and pose a serious threat to the agricultural industry and food security. Plant growth-promoting endophytic bacteria Bacillus subtilis are considered as a bioactive and eco-friendly strategy for plant protection. Earlier, we have shown B. subtilis 10-4 has a growth-promoting and anti-stress effect on wheat under water deficiency. Here, we investigated the effect of B. subtilis 10-4 and B. subtilis 10-4+salicylic acid (SA) on growth and tolerance of wheat (cv. ‘Omskaya-35’) to combined drought (12%PEG) and Fusarium culmorum. 12%PEG and F. culmorum led to yellowing of leaves (in addition to traces of the root damages). Inoculation with 10-4 and especially 10-4+SA reduced the fusarium development in wheat under drought. Similar effects were revealed for growth parameters. Also, 10-4 (especially 10-4+SA) reduces stress-induced lipid peroxidation (MDA). Such physiological effect may be connected with the ability of strain 10-4 to colonize the internal tissues of host-plant and regulate metabolism from the inside. The obtained construct based on the plasmid pHT01 and the green fluorescent protein (gfp) gene, by which was modified the strain 10-4, will allow revealing the nature of the symbiotic relationships between the strain 10-4 and host-plant. The findings indicate that application B. subtilis 10-4 and its composition with SA may be an effective strategy to increase wheat tolerance to the combined abiotic/biotic stresses.

Global food demand is expected to be doubled by 2050, while natural resources are continuously under threat due to unpredictable climatic changes. This challenge can be tackled by increasing the yield of the crops and by reducing abiotic stresses such as water stress. Research shows that due to water stress the morphology and the structure of plant’s canopy changes. The first step in building early water stress detection system is to extract accurate area where photosynthetic activities of the plant are occurring. In this research work, comparative analysis of seven different segmentation algorithms viz., convolution gradient-based, watershed, mean-shift, k-means, Global static thresholding, Otsu thresholding and hybrid approach (combination of Global Static thresholding with k-means) has been analyzed in order to identify the most probable area of canopy where maximum photosynthetic signals can be captured. The comparison is done in terms of IoU metric. The comparative results indicate that the most appropriate method for wheat canopy segmentation is a hybrid approach, which achieves IoU score of 59.8 and its runner up algorithm is Global Static Thresholding with an IoU score 53.8.

Drought and other abiotic stresses have major negative effects on agricultural productivity. The plant hormone abscisic acid (ABA) regulates many responses to environmental stresses and can be used to improve crop performance under stress. ABA levels rise in response to diverse abiotic stresses to coordinate physiological and metabolic responses that help plants survive stressful environments. In all land plants, ABA receptors are responsible for initiating a signaling cascade that leads to stomata closure, growth arrest and large-scale changes in transcript levels required for stress tolerance. We wanted to test the meaning of root derived ABA signaling in drying soil on water balance. To this end we generated transgenic tomato lines in which ABA signaling is initiated by a synthetic agonist- mandipropamid. Initial study using a Series of grafting experiments indicate that that root ABA signaling has no effect on the immediate regulation of stomata aperture. Once concluded, these experiments will enable us to systematically dissect the physiological role of root-shoot interaction in maintaining the water balance in plants and provide new tools for targeted improvement of abiotic stress tolerance in crop plants.

Major threats to agricultural sustainability in the 21st century are drought, increasing temperatures, soil salinity and soilborne pathogens, all of which are being exacerbated by climate change and pesticide abolition and are burning issues related to agriculture in the Middle East. We have found that Class 2 fungal endophytes adapt native plants to environmental stresses (drought, heat and salt) in a habitat-specific manner, and that these endophytes can confer stress tolerance to genetically distant monocot and eudicot hosts. In the past, we generated a uv non-pathogenic endophytic mutant of Colletotrichum magna (path-1) that colonized cucurbits, induced drought tolerance and enhanced growth, and protected 85% - 100% against disease caused by certain pathogenic fungi. We propose: 1) utilizing path-1 and additional endophtyic microorganisms to be isolated from stress-tolerant local, wild cucurbit watermelon, Citrulluscolocynthis, growing in the Dead Sea and Arava desert areas, 2) generate abiotic and biotic tolerant melon crop plants, colonized by the isolated endophytes, to increase crop yields under extreme environmental conditions such as salinity, heat and drought stress, 3) manage soilborne fungal pathogens affecting curubit crop species growing in the desert areas. This is a unique and novel "systems" approach that has the potential to utilize natural plant adaptation for agricultural development. We envisage that endophyte-colonized melons will eventually be used to overcome damages caused by soilborne diseases and also for cultivation of this crop, under stress conditions, utilizing treated waste water, thus dealing with the limited resource of fresh water.

Plants emit a bouquet of volatile organic compounds (VOCs) in response to both biotic and abiotic stresses and, simultaneously, eavesdrop on emit-ted signals to activate direct and indirect defenses. By gaining even a slight insight into the semantics of interplant communications, a unique aware-ness of the operational environment may be obtainable (e.g., knowledge of a disturbance within). In this effort, we used five species of plants, Arabidopsis thaliana, Panicum virgatum, Festuca rubra, Tradescantia zebrina, and Achillea millefolium, to produce and query VOCs emitted in response to mechanical wounding and light cycles. These plants provide a basis for further investigation in this communication system as they span model organisms, common house plants, and Arctic plants. The VOC com-position was complex; our parameter filtering often enabled us to reduce the noise to fewer than 50 compounds emitted over minutes to hours in a day. We were able to detect and measure the plant response through two analytical methods. This report documents the methods used, the data collected, and the analyses performed on the VOCs to determine if they can be used to increase environmental awareness of the battlespace.

Based on the type of relationship with the host, plant viruses can be grouped as acute or persistent. Acute viruses are well studied and cause disease. In contrast, persistent viruses do not appear to affect the phenotype of the host. The genus Endornavirus contains persistent viruses that infect plants without causing visible symptoms. Infections by endornaviruses have been reported in many economically important crops, such as avocado, barley, common bean, melon, pepper, and rice. However, little is known about the effect they have on their plant hosts. The long term objective of the proposed project is to elucidate the nature of the symbiotic interaction between Bell pepper endornavirus (BPEV) and its host. The specific objectives include: a) to evaluate the phenotype and fruit yield of endornavirus-free and endornavirus-infected bell pepper near-isogenic lines under greenhouse conditions; b) to conduct gene expression studies using endornavirus-free and endornavirus-infected bell pepper near-isogenic lines; and c) to study the interactions between acute viruses, Cucumber mosaic virus Potato virus Y, Pepper yellow leaf curl virus, and Tobacco etch virus and Bell pepper endornavirus. It is likely that BPEV in bell pepper is in a mutualistic relationship with the plant and provide protection to unknown biotic or abiotic agents. Nevertheless, it is also possible that the endornavirus could interact synergistically with acute viruses and indirectly or directly cause harmful effects. In any case, the information that will be obtained with this investigation is relevant to BARD’s mission since it is related to the protection of plants against biotic stresses.

In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.

General The final goal and overall objective of the current research has been to modify lipid hydroperoxidation in order to create desirable phenotypes in two important crops, potato and tomato, which normally are exposed to abiotic stress associated with such oxidation. The specific original objectives were: (i) the roles of lipoxygenase (LOX) and phospholipids hydroperoxide glutathione peroxidase (PHGPx) in regulating endogenous levels of lipid peroxidation in plant tissues; (ii) the effect of modified lipid peroxidation on fruit ripening, tuber quality, crop productivity and abiotic stress tolerance; (iii) the effect of simultaneous reduction of LOX and increase of PHGPx activities on fruit ripening and tuber quality; and (iv) the role of lipid peroxidation on expression of specific genes. We proposed to accomplish the research goal by genetic engineering of the metabolic activities of LOX and PHGPx using regulatable and tissue specific promoters, and study of the relationships between these two consecutive enzymes in the metabolism and catabolism of phospholipids hydroperoxides. USA Significant progress was made in accomplishing all objectives of proposed research. Due to inability to regenerate tomato plants after transforming with 35S-PHGPx chimeric gene construct, the role of low catalase induced oxidative stress instead of PHGPx was evaluated on agronomical performance of tomato plant and fruit quality attributes. Effects of polyamine, that protects DNA from oxidative stress, were also evaluated. The transgenic plants under expressing lipoxygenase (LOX-sup) were crossed with catalase antisense (CAT-anti) plants or polyamine over producing plants (SAM-over) and the lines homozygous for the two transgenes were selected. Agronomical performance of these line showed that low catalase induced oxidative stress negatively affected growth and development of tomato plants and resulted in a massive change in fruit gene expression. These effects of low catalase activity induced oxidative stress, including the massive shift in gene expression, were greatly overcome by the low lipoxygenase activity. Collectively results show that oxidative stress plays significant role in plant growth including the fruit growth. These results also for the first time indicated that a crosstalk between oxidative stress and lipoxygenase regulated processes determine the outcome during plant growth and development. Israel Regarding PHGPx, most of the study has concentrated on the first and the last specific objectives, since it became evident that plant transformation with this gene is not obvious. Following inability to achieve efficient transformation of potato and tomato using a variety of promoters, model plant systems (tobacco and potato cell cultures, tobacco calli and plantlets, and Arabidopsis) were used to establish the factors and to study the obstacles which prohibited the regeneration of plants carrying the genetic machinery for overproduction of PHGPx. Our results clearly demonstrate that while genetic transformation and over-expression of PHGPx occurs in pre-developmental tissue stage (cell culture, calli clusters) or in completed plant (Arabidopsis), it is likely that over-expression of this enzyme before tissue differentiation is leading to a halt of the regeneration process. To support this assumption, experiments, in which genetic engineering of a point-mutated PHGPx gene enable transformation and over-expression in plants of PhSPY modified in its catalytic site and thus inactive enzymatically, were successfully carried out. These combined results strongly suggest, that if in fact, like in animals and as we established in vitro, the plant PHGPx exhibits PH peroxidase activity, these peroxides are vital for the organisms developmental process.

The main objective of the proposed research was to study IQD1's mechanism of action and elucidate its role in plant protection. Preliminary experiments suggest that IQD1 binds CaM in a Ca²⁺-dependent manner and functions in general defense responses. We propose to identify proteins and genes that interact with IQD1, which may provide some clues to its mechanism of action. We also plan to dissect IQD1's integration in defense pathways and to study and modulate its binding affinity to CaM in order to enhance crop resistance. Our specific objectives were: (1) Analysis of IQD1's CaM-binding properties; (2) Identification of IQD1 targets;(3) Dissection of IQD1 integration into defense signaling pathways. Analysis of IQD1's CaM-binding properties defined four potential classes of sequences that should affect CaM binding: one is predicted to raise the affinity for Ca²⁺-dependent interaction but have no effect on Ca²⁺-independent binding; a second is predicted to act like the first mutation but eliminate Ca²⁺-independent binding; a third has no predicted effect on Ca²⁺-dependent binding but eliminates Ca²⁺-independent binding; and the fourth is predicted to eliminate or greatly reduce both Ca²⁺-dependent and Ca²⁺-independent binding. Following yeast two hybrid analysis we found that IQD1 interact with AtSR1 (Arabidopsis thalianaSIGNALRESPONSIVE1), a calcium/calmodulin-binding transcription factor, which has been shown to play an important role in biotic and abiotic stresses. We tested IQD1 interaction with both N-terminal or C-terminal half of SR1. These studies have uncovered that only the N-terminal half of the SR1 interacts with the IQD1. Since IQD1 has an important role in herbivory, its interaction with SR1 suggests that it might also be involved in plant responses to insect herbivory. Since AtSR1, like IQD1, is a calmodulin-binding protein and the mutant showed increased sensitivity to a herbivore, we analyzed WT, Atsr1 and the complemented line for the levels of GS to determine if the increased susceptibility of Atsr1 plants to T. ni feeding is associated with altered GS content. In general, Atsr1 showed a significant reduction in both aliphatic and aromatic GS levels as compared to WT. In order to study IQD1's molecular basis integration into hormone-signaling pathways we tested the epistatic relationships between IQD1 and hormone-signaling mutants. For that purpose we construct double mutants between IQD1ᴼXᴾ and mutants defective in plant-hormone signaling and GS accumulation. Epitasis with SA mutant NahG and npr1-1 and JA mutant jar1-1 suggested IQD1 function is dependent on both JA and SA as indicated by B. cinerea infection assays. We also verified the glucosinolate content in the crosses siblings and found that aliphatic GSL content is reduced in the double transgenic plants NahG:IQD1ᴼXᴾ as compare to parental lines while the aliphatic GSL content in the npr1-1:IQD1ᴼXᴾ and jar1-1: IQD1ᴼXᴾ double mutants was intimidated to the parental lines. This suggests that GSL content dependency on SA is downstream to IQD1. As a whole, this project should contribute to the development of new defense strategies that will improve crop protection and reduce yield losses and the amount of pesticides required; these will genuinely benefit farmers, consumers and the environment.

Plant yield largely depends on a complex interplay and feedback mechanisms of distinct hormonal pathways. Over the past decade great progress has been made in elucidating the global molecular mechanisms by which each hormone is produced and perceived. However, our knowledge of how interactions between hormonal pathways are spatially and temporally regulated remains rudimentary. For example, we have demonstrated that although the BR receptor BRI1 is widely expressed, the perception of BRs in epidermal cells is sufficient to control whole-organ growth. Supported by additional recent works, it is apparent that hormones are acting in selected cells of the plant body to regulate organ growth, and furthermore, that local cell-cell communication is an important mechanism. In this proposal our goals were to identify the global profile of translated genes in response to BR stimulation and depletion in specific tissues in Arabidopsis; determine the spatio-temporal dependency of BR response on auxin transport and signaling and construct an interactive public website that will provide an integrated analysis of the data set. Our technology incorporated cell-specific polysome isolation and sequencing using the Solexa technology. In the first aim, we generated and confirmed the specificity of novel transgenic lines expressing tagged ribosomal protein in various cell types in the Arabidopsis primary root. We next crossed these lines to lines with targeted expression of BRI1 in the bri1 background. All lines were treated with BRs for two time points. The RNA-seq of their corresponding immunopurified polysomal RNA is nearly completed and the bioinformatic analysis of the data set will be completed this year. Followed, we will construct an interactive public website (our third aim). In the second aim we started revealing how spatio-temporalBR activity impinges on auxin transport in the Arabidopsis primary root. We discovered the unexpected role of BRs in controlling the expression of specific auxin efflux carriers, post-transcriptionally (Hacham et al, 2012). We also showed that this regulation depends on the specific expression of BRI1 in the epidermis. This complex and long term effect of BRs on auxin transport led us to focus on high resolution analysis of the BR signaling per se. Taking together, our ongoing collaboration and synergistic expertise (hormone action and plant development (IL) and whole-genome scale data analysis (US)) enabled the establishment of a powerful system that will tell us how distinct cell types respond to local and systemic BR signal. BR research is of special agriculture importance since BR application and BR genetic modification have been shown to significantly increase crop yield and to play an important role in plant thermotolerance. Hence, our integrated dataset is valuable for improving crop traits without unwanted impairment of unrelated pathways, for example, establishing semi-dwarf stature to allow increased yield in high planting density, inducing erect leaves for better light capture and consequent biomass increase and plant resistance to abiotic stresses.

The overall objective of this research was to elucidate the horticultural, pathological, physiological and molecular factors impacting melon varieties (scion) grafted onto M. cannonballus resistant melon and squash rootstocks. Specific objectives were- to compare the performance of resistant melon germplasm (grafted and non-grafted) when exposed to M. cannoballus in the Lower Rio Grande valley and the Wintergarden, Texas, and in the Arava valley, Israel; to address inter-species relationships between a Monosporascus resistant melon rootstock and susceptible melon scions in terms of fruit-set, fruit quality and yield; to study the factors which determine the compatibility between the rootstock and the scion in melon; to compare the responses of graft unions of differing compatibilities under disease stress, high temperatures, deficit irrigation, and salinity stress; and to investigate the effect of rootstock on stress related gene expression in the scion. Some revisions were- to include watermelon in the Texas investigations since it is much more economically important to the state, and also to evaluate additional vine decline pathogens Didymella bryoniae and Macrophomina phaseolina. Current strategies for managing vine decline rely heavily on soil fumigation with methyl bromide, but restrictions on its use have increased the need for alternative management strategies. Grafting of commercial melon varieties onto resistant rootstocks with vigorous root systems is an alternative to methyl bromide for Monosporascus root rot/vine decline (MRR/VD) management in melon production. Extensive selection and breeding has already produced potential melon rootstock lines with vigorous root systems and disease resistance. Melons can also be grafted onto Cucurbita spp., providing nonspecific but efficient protection from a wide range of soil-borne diseases and against some abiotic stresses, but compatibility between the scion and the rootstock can be problematic. During the first year experiments to evaluate resistance to the vine decline pathogens Monosporascus cannonballus, Didymella bryoniae, and Macrophomina phaseolina in melon and squash rootstocks proved the efficacy of these grafted plants in improving yield and quality. Sugars and fruit size were better in grafted versus non-grafted plants in both Texas and Israel. Two melons (1207 and 124104) and one pumpkin, Tetsukabuto, were identified as the best candidate rootstocks in Texas field trials, while in Israel, the pumpkin rootstock RS59 performed best. Additionally, three hybrid melon rootstocks demonstrated excellent resistance to both M. cannonballus and D. bryoniae in inoculated tests, suggesting that further screening for fruit quality and yield should be conducted. Experiments with ABA in Uvalde demonstrated a significant increase in drought stress tolerance and concurrent reduction in transplant shock due to reduced transpiration for ‘Caravelle’ plants. In Israel, auxin was implicated in reducing root development and contributing to increased hydrogen peroxide, which may explain incompatibility reactions with some squash rootstocks. However, trellised plants responded favorably to auxin (NAA) application at the time of fruit development. Gene expression analyses in Israel identified several cDNAs which may code for phloem related proteins, cyclins or other factors which impact the graft compatibility. Manipulation of these genes by transformation or traditional breeding may lead to improved rootstock cultivars. Commercial applications of the new melon rootstocks as well as the ABA and TIBA growth regulators have potential to improve the success of grafted melons in both Israel and Texas. The disease resistance, fruit quality and yield data generated by the field trials will help producers in both locations to decide what rootstock/scion combinations will be best.

The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.