Dissertations / Theses on the topic 'Plants Metabolism'

In this study, the biosynthesis of albizziine has been elucidated, and a direct precursor relationship shown to exist between uracil and albizziine. This was confirmed by the demonstration that [2-¹⁴C]uracil specifically labels C-5 of albizziine. It is concluded that the biosynthetic sequence involves the hydroxylation of uracil to isobarbituric acid, then amination to 5-aminouracil, followed by hydrogenation and ring-opening, to yield albizziine. 2,3-Diaminopropanoic acid was shown to be formed from albizziine by the action of β-ureidopropionase. Thus, the formation of albizziine and 2,3-diaminopropanoic acid represents a further aspect of the interfacing of pyrimidine primary and secondary metabolism through uracil. Lathyrine was shown to be catabolyzed in Lathyrus tingitanus to yield the non-protein amino acid 4-hydroxyhomoarginine, and it was thus confirmed that 4-hydroxyhomoarginine is a catabolite rather than a precursor of lathyrine. 2-Amino-4-carboxypyrimidine, the immediate precursor of the lathyrine ring-system, was shown to be synthesized enzymically from uracil. The relative amount of exogenously supplied uracil diverted into production of the isomeric pyrimidinyl amino acids willardiine and isowillardiine in Pisum sativum, and also that diverted into the production of the pyrimidine amino acid lathyrine in Lathyrus tingitanus was determined. Uracil was shown to have a pronounced inhibitory effect on the germination and growth of Phaseolus aureus and Glycine max. As these plants do not produce pyrimidine-derived secondary products, this observation is consistent with the view that production of such compounds is a detoxification mechanism for bioactive pyrimidines.

Thesis (Ph. D.)--University of Kentucky, 2004.
Title from document title page (viewed Jan. 7, 2005). Document formatted into pages; contains x, 127p. : ill. Includes abstract and vita. Includes bibliographical references (p. 118-126).

This research explored fate of organic contaminants in aquatic plant systems through (i) experimental development of relationships to describe sorption, uptake and enzymatic processing of contaminants by plants and inhibition of aquatic plants by contaminants and (ii) incorporation of experimental relationships into a conceptual model which describes contaminant fate in aquatic plant systems. This study focused on interactions of aquatic plants L. minor and M. aquaticum with halogenated phenols. 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4-dichlorophenol (2,4-DCP) are precursors for the highly toxic and heavily applied herbicides 2,4,5-T and 2,4-D and were examined in detail. Chlorophenols are generally resistant to microbial degradation, a property which may limit microbial remediation options as effective alternatives for clean up of contaminated sites. Relationships for fundamental interactions between plants and contaminants that dictate uptake, enzymatic processing and sequestration of contaminants by aquatic plants were established. An assay which quantified production of oxygen by plants was developed to quantify plant metabolic activity and inhibition. Uptake of chlorinated phenols depended on plant activity and aqueous phase concentration of contaminant in the protonated form. Therefore, plant activity, contaminant pKa and media pH were established as critical parameters controlling rate of contaminant uptake. A conceptual model was developed which incorporated plant activity and inhibition into a mathematical description of uptake of organic contaminants by aquatic plants. The conceptual model was parameterized using experimental data delineating effect of plant activity, inhibition and speciation on contaminant uptake and the model was verified using independently gathered data. Experimentation with radio-labeled chlorinated phenols established that contaminants were sequestered internal to plants by plant enzymatic processing. 19F NMR was established as a technique to quantify transformation and conjugation products internal to plants and contaminant assimilation by plants and demonstrated that multiple metabolites containing the parent compound were present and quantifiable internal to plants. Finally, fate of plant-sequestered contaminants in an anaerobic bioassay was examined using Desulfitobacterium sp. strain Viet1. The results of this study address the role of aquatic plants in sequestration of contaminants in surface waters that indicate the potential and limitations of use of aquatic plants in natural and engineered treatment systems.

The aim of this thesis was to develop stable-isotope steady-state metabolic flux analysis (MFA) based on ¹³C labeling to quantify intracellular fluxes of central carbon metabolism in plants. The experiments focus on the analysis of a heterotrophic cell suspension culture of Arabidopsis thaliana (L) Heynh. (ecotype Landsberg erecta). The first objective was to develop a robust methodology based on combining high quality steady-state stable labeling data, metabolic modeling and computational analysis. A comprehensive analysis of the factors that influence the outcome of MFA was undertaken and best practice established. This allowed a critical analysis of the subcellular compartmentation of carbohydrate oxidation in the cell culture. The second objective was to apply the methodology to nutritional perturbations of the cell suspension. A comparison of growth on different nitrogen sources revealed that transfer to an ammonium-free medium: (i) increased flux through the oxidative pentose phosphate pathway (oxPPP) by 10% relative to glucose utilisation; (ii) caused a substantial decrease in entry of carbon into the tricarboxylic acid cycle (TCA); and (iii) increased the carbon conversion efficiency from 55% to 69%. Although growth on nitrate alone might be expected to increase the demand for reductant, the cells responded by decreasing the assimilation of inorganic N. Cells were also grown in media containing different levels of inorganic phosphate (Pi). Comparison of the flux maps showed that decreasing Pi availability: (i) decreased flux through the oxPPP; (ii) increased the proportion of substrate fully oxidised by the TCA cycle; and (iii) decreased carbon conversion efficiency. These changes are consistent with redirection of metabolism away from biosynthesis towards cell maintenance as Pi is depleted. Although published genome-wide transcriptomic and metabolomic studies suggest that Pi starvation leads to the restructuring of carbon and nitrogen metabolism, the current analysis suggests that the impact on metabolic organisation is much less extreme.

The biosynthesis of ABA from R-[2-¹⁴C]-MVA was demonstrated in Persea americana cv. Fuerte mesocarp and in mature seeds of Hordeum vulgare cv. Dyan and cv. Himalaya. Radioactivity from R-[2-¹⁴-C]-MVA was also incorporated into the 1',4'-trans ABA diol in Persea americana mesocarp and a possible role for this metabolite as a precursor of ABA in plants is discussed. The biosynthesis of ABA from MVA could not be demonstrated in either turgid and waterstressed Hordeum vulgare cv. Dyan, Pisum sativum cv. Black-eyed Susan and Phaseolus vulgaris cv. Top-crop or in immature seeds of Pisum sativum and Phaseolus vulgaris. (R,S,)-[2-¹⁴C]-ABA was catabolised to PA, DPA and aqueous conjugates in leaves and mature seeds of Hordeum vulgare cv. Dyan, seedlings and immature seeds of Pisum sativum and Phaseolus vulgaris and in mesocarp from ripening fruits of Persea americana. PA and DPA were identified by either microchemical methods and/or capillary GC-MS. 7'-Hydroxy ABA was characterised as a novel ABA catabolite in light-grown and etiolated leaves of Hordeum vulgare by capillary GC-MS. Circular dichroism analysis revealed that it was derived predominantly from the (R)-enantiomer of ABA. This catabolite was absent in similar studies using the dicotyledons Pisum sativum and Phaseolus vulgaris. Refeeding studies with [¹⁴C]-PA, [C]-DPA and [¹⁴C]-7'-hydroxy ABA were used to confirm the metabolic interrelationship between ABA and its catabolites in both vegetative and non-vegetative tissues from monocotyledonous and dicotyledonous species. The methyl ester of (R,S,)-ABA was hydrolysed efficiently by light-grown leaves of Hordeum vulgare. Older, vegetative tissues catabolised (R,S,)-ABA more efficiently than their younger counterparts. In contrast, small, immature seeds of Pisum sativum catabolised (R,S,)-ABA more effectively than larger, immature seeds of this species. Light did not appear to influence ABA biosynthesis but markedly enhanced ABA catabolism. Light stimulated the overall rate of ABA catabolism in both vegetative and non-vegetative tissue. Water stress reduced ABA catabolism in Hordeum vulgare leaves but had little effect on this process in Phaseolus vulgaris seedlings. Pretreatment of tissues with (R,S,)-ABA retarded the catabolism of (R,S,)-[2-¹⁴C]-ABA, negating ABA-induced conversion to PA. Cycloheximide inhibited ABA biosynthesis and catabolism but did not affect ABA conjugation. Chloramphenicol and lincomycin had little or no effect on ABA metabolism suggesting that the enzymes involved were labile and cytoplasmic in origin. Ancymidol and cycocel inhibited ABA biosynthesis while AM01618 stimulated this process. The cytokinins, benzyladenine, kinetin, isopentenyl adenine and zeatin also inhibited ABA biosynthesis. These results are discussed in relation to the possible involvement of carotenoids in ABA biosynthesis. AM01618, ancymidol andcycocel did not significantly influence the conversion of ABA to PA and DPA while cytokinins appeared to enhance this process only in vegetative tissue. The information derived from these studies was then used in attempts to develop a cell-free system from higher plants capable of metabolising ABA. A cell-free system prepared from imbibed Hordeum vulgare cv. Dyan embryos biosynthesized and catabolised ABA. This is the first demonstration of a cell-free system from non-vegetative tissue capable of metabolising ABA and could prove useful for elucidating its biosynthetic route. This cell-free system generated the terpenyl pyrophosphates IPP, FPP and GGPP from MVA. ABA was produced from both MVA and IPP in the presence of 0₂ and NADPH. The biosynthesis of ABA was stimulated by the addition of the squalene 2,3-oxide cyclase and kaurene synthetase inhibitor, AM01618 and a "cold-pool trap" of (R,S,)-ABA. Ancymidol, cycocel and cytokinins reduced incorporation of label from MVA into ABA. Similar cell-free preparations, in the absence of AM01618, converted (R,S,)-[2-¹⁴-C]-ABA into PA, 7'-hydroxy ABA and water-soluble conjugates. Although the methyl ester of (R,S,)-ABA was efficiently hydrolysed in this cell-free system no DPA was generated. The possible involvement of mixed function oxidase activity and soluble oxidases is discussed in relation to ABA metabolism. While cell-free preparations from Persea americana cv. Fuerte mesocarp and immature seeds of Pisum sativum and Phaseolus vulgaris were unable to synthesize ABA from MVA, these tissue homogenates converted ABA into more polar acidic products. PA and DPA were identified as products of ABA catabolism in extracts from immature seeds of Phaseolus vulgaris and the l',4'-cis diol of ABA in extracts from Pisum sativum immature seeds

Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq)
Os terpenos constituem a classe de produtos naturais com maior diversidade química e estrutural, estando associados ao metabolismo das plantas e às interações destas com outros organismos. Estes compostos, as enzimas que os sintetizam e as plantas que os produzem são amplamente estudados em diferentes aspectos. Para melhor compreensão da expressão de terpenos em plantas sob estresse biótico, as espécies Varronia curassavica e Pistacia palaestina foram estudadas neste trabalho. Folhas de V. curassavica contém óleo essencial rico em sesquiterpenos com propriedades anti-inflamatórias, especialmente α-humuleno and β-cariofileno. O objetivo deste estudo foi avaliar as respostas do seu metabolismo em função da aplicação de dois eliciadores naturais no conteúdo de sesquiterpenos de V. curassavica. Para isso, as plantas receberam a aplicação de acibenzolar-S-metil (500 mg L-1), 1,6 β-D-glucano obtido a partir de Lasiodiplodia theobromae (50 mg L-1) e água destilada como controle, sendo realizada as avaliações de trocas gasosas, atividade das enzimas fenilalanina amônia-liase, superóxido dismutase, peroxidase e catalase, bem como a análise química do óleo essencial. Acibenzolar-S-metílico reduziu significativamente a taxa líquida de assimilação de carbono e a concentração intercelular de CO2, enquanto que 1,6 β-D-glucano reduziu significativamente apenas a concentração intercelular de CO2. O maior rendimento de óleo essencial (0.819%) foi obtido em plantas eliciadas por 1,6 β-D-glucano. As proporções relativas e a quantidade de α-humuleno e β-cariofileno não diferiram entre os tratamentos, porém os eliciadores aumentaram significativamente a atividade da enzima guaiacol peroxidase. Os terpenos estão presentes nas folhas e galhas de Pistacia palaestina. Apesar do mecanismo de desenvolvimento das galhas ainda não ter sido completamente elucidado, sabe-se que espécies de afídeos como Baizongia pistaciae L. manipulam anatomicamente, fisiologicamente e quimicamente as plantas hospedeiras em seu benefício. Por este motivo, o isolamento e a caracterização funcional dos genes que codificam terpeno sintases em galhas induzidas por B. pistaciae, bem como sua expressão relativa por RT-qPCR em folhas e galhas de P. palaestina foram os objetivos deste trabalho. A expressão heteróloga foi realizada em E. coli pLYS-BL21, sendo as reações enzimáticas feitas com proteínas purificadas e usando geranil difosfato (GPP) ou farnesil difosfato (FPP) para testar a atividade das enzimas como mono- e sesquiterpeno sintases, respectivamente. Para o experimento de RT-qPCR, foi selecionado um gene referência entre actina, ciclofilina, fosfoglicerato quinase, RNA polimerase II, α-tubulina e ubiquitina. Em seguida, realizaram-se reações com as terpeno sintases para avaliar as diferenças nos níveis de expressão em folhas não colonizadas e galhas. Foram isoladas e caracterizadas duas monoterpeno sintases (PpTPS281 e PpTPS809) e uma sesquiterpeno sintase (PpTPS232) em P. palaestina. PpTPS281 produziu exclusivamente D-limoneno a partir de GPP, enquanto PpTPS809 produziu vários monoterpenos a partir de GPP e PpTPS232 catalisou a formação de diferentes sesquiterpenos a partir de FPP. Os resultados da RT-qPCR mostraram que o gene actina é o mais apropriado para ser usado na comparação de expressão de genes de folhas não colonizadas e galhas induzidas por B. pistaciae. Os níveis de expressão dos três genes foram significativamente aumentados em galhas (de 2,21- a 96.5-vezes), quando comparados com folhas.
Terpenes are a large and diverse class of natural products, being associated with plant metabolism and interactions with other organisms. Nowadays compounds and enzymes of the terpenes pathway in plants are widely studied in different aspects. Varronia curassavica and Pistacia palaestina were the selected species to study the biotic stress effects on terpenoids expressions. Leaves of V. curassavica are the commercial source α-humulene and β-caryophyllene, sesquiterpenes with anti-inflammatory properties. The objective of this study was to evaluate the effect of two natural elicitors on the sesquiterpene content of V. curassavica. Thus, field grown plants received the application of acibenzolar-S-methyl (500 mg L-1), 1,6 -D-glucan obtained from Lasiodiplodia theobromae (50 mg L-1) and distilled water as a control. Gas exchange rate, terpene enzymes such as phenylalanine ammonia-lyase, superoxide dismutase, guaiacol peroxidase and catalase activity and essential oil content in leaves were measured. Acibenzolar-S-methyl reduced significantly the net carbon assimilation rate and the intercellular CO2 concentration, while1,6 -D-glucan reduced significantly only the intercellular CO2 concentration. The highest essential oil yield (0.819%) was obtained in plants elicited with 1,6 -D-glucan. The content of α-humulene and β-caryophyllene did not differ among treatments however the elicitors provided a significant increase in guaiacol peroxidase activity. Terpenes are present in Pistacia palaestina in leaves and in horn-shaped galls. The mechanism of gall development remains unknown, however it is clear Baizongia pistaciae L., an aphid species, manipulates their hosts anatomy, physiology, and chemistry for their own benefit. To isolate and functional characterize terpene synthase genes from galls induced by B. pistaciae as well as their gene relative expression by RT-qPCR in leaves and galls of P. palestina were the aims of this study. The heterologous expression was performed in E. coli pLYS-BL21 cells, being enzymatic assay reactions made with the purified proteins using geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) to test for possible mono- and sesquiterpene synthase activity, respectively. For relative real-time PCR, it was selected an appropriate reference gene between actin, cyclophilin, phosphoglycerate kinase, RNA polymerase II, α-tubulin and ubiquitin. After selection, it was performed reactions with terpene synthases genes to evaluate differences in expression levels in P. palaestina non-colonized leaves and galls. Two monoterpene synthases (PpTPS281 and PpTPS809) and one sesquiterpene synthase (PpTPS232) were isolated and characterized in P. palaestina. PpTPS281 produced exclusively D-limonene from GPP, while PpTPS809 produced several monoterpenes from GPP and PpTPS232 catalyzed the formation of different sesquiterpenes from FPP. Real-time PCR results showed that actin is the most proper gene to be used for genes expression studies between non-colonized P. palaestina leaves and galls induced by B. pistaciae. The levels of expression of the genes were significantly upregulated in galls (from 2,21- to 96.5-fold) when compared to leaves.

A utilização de plantas na cura dos mais diversos males é tradicionalmente conhecida e utilizada há centenas de anos pela humanidade. As células vegetais produzem uma grande variedade de compostos químicos entre o metabolismo primário e secundário. Os produtos do metabolismo secundário são de especial interesse, pois são responsáveis por diversas ações biológicas. Informações etnobotânicas indicam que mais de 800 plantas são usadas como remédios tradicionais para o tratamento da diabetes. Muitas espécies pertencentes ao gênero Salacia têm sido descritas com atividade antidiabética, entre elas Salacia impressifolia. É uma espécie pertencente à família Celastraceae, conhecida popularmente no Brasil como miraruíra. Este trabalho visou desvendar a composição química desta espécie, utilizando para isso extratos obtidos a partir da casca e do caule. Em seguida, realizou-se análise química dos extratos por espectrometria de massas de alta resolução (EMAR). Nestes foram identificados 32 compostos, os quais haviam sido previamente descritos na literatura para o mesmo gênero. Entre eles destacam-se o kotalanol, salacinol e o mangiferina, com ação hipoglicemiante previamente descrita por outros autores. Para análise in vivo do potencial hipoglicemiante, realizou-se ensaio com o extrato hidroalcoólico do caule da planta em ratos com diabetes induzida por estreptozotocina. O extrato apresentou ação semelhante ao fármaco controle glibenclamida. Também foi avaliado o perfil lipídico (triglicerídeos, colesterol total e HDL) e as enzimas hepáticas TGO e TGP, onde pode-se observar aumento de HDL.
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The use of plants in various ailments healing is traditionally known and used for centuries by mankind. Plant cells produce a large variety of chemical compounds between the primary and secondary metabolism. The secondary metabolism products are of special interest, as they are responsible for several biological activities. Ethnobotanic information indicates that more than 800 plants are used as traditional remedies in the treatment of diabetes. Many species of Salacia genus have been described with antidiabetic activity, including Salacia impressifolia. This specie belongs to Celastraceae family, popularly known as miraruíra in Brazil. This work aimed to develop the chemical composition of this specie through stem and stem bark extracts. Chemical analysis of the extracts was carried out by high resolution mass spectrometry (HRMS). In these extracts, 32 compounds were identified, previously described for the same genus. Among them, were identified kotalanol, salacinol and mangiferina, with hypoglycemic action previously described by others authors. For in vivo analysis of hypoglycemic activity, it was performed the hydroalcoholic stem extract in streptozotocin induced diabetics rats. The extract presented similar action to control drug glibenclamide. It was also assessed the lipid profile (triglycerides, total cholesterol and HDL) and liver enzymes AST and ALT, and it was observed an increase in HDL level.

Dissertation (PhD) -- University of Stellenbosch, 2004.
ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena.
AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer om suikerverspreiding te koordineer in reaksie op beide inherente- en omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die modulering van geenuitdrukking. Die genetiese transformasie van plante en die gevolglike daarstelling van transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese deur suikers, die belang van kompartementalisering ten opsigte van sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in floëemontlaaiing en swelgpuntkrag. In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in hierdie plante te bepaal. Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter, NtMst1, het beduidend toegeneem in die bron-blare en wortels van die Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking het afgehang van die subsellulêre kompartement waarin die gis-invertase uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is, met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia, bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele beheer deur suikers 'n belangrike rol in die regulering van hierdie proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne, dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van die swelgpuntkrag van 'n swelgpuntorgaan kan wees. Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo- Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies, asook die belang van invertases in die regulering van suikerverdeling gegewe die vermoë om swelgpuntkrag te verander. Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie bekragtig die belang van rekombinante DNA tegnologie in die bestudering van fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt interaksies in hierdie lyne met die gevolglike ontginning van waardevolle inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op die komplekse en aanpasbare aard van die verhouding wat bestaan tussen suikermetabolisme, -verdeling en groeiverskynsels.

Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules.

The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar.

Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database.

The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species.

Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves).

By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants.

Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.

NO2 is a common atmospheric pollutant. It can enter plants through leaf absorption and participate in general N metabolism to ameliorate N-deficiency in plants. However, NO2 exposure sometimes exerts detrimental effects on plant growth. The hypotheses to explain the mechanism of these detrimental effects have remained inconsistent. Besides formation of nitrate and nitrite as an additional N supply to plants, NO2 absorption by leaves also brings H+ into plants. Almost every process of nitrogen uptake and assimilation, (such as uptake of nitrate and ammonium, fixation of N2, absorption of NO2, reduction of nitrate and nitrite, and synthesis of amino acids from ammonium), involves production or consumption of H+. Therefore NO2 absorption and N metabolism may interfere with each other through changes in concentration of H+, which take part in reactions of N metabolism and influence activity of the enzymes catalyzing biochemical reactions in N metabolism of plants. This study aims to specify the effects of NO2 exposure on nitrogen uptake and metabolism in soybean plants, and to explore the mechanistic links between the acidity changes caused by NO2 absorption and N metabolism, and the effects of NO2 on N metabolism and growth of plants. The study was conducted mainly through exposure experiments. Plants were supplied with ammonium or 15N-labelled nitrate, and exposed to NO2 or clean air. H+ concentrations and concentrations of the products of N uptake and metabolism in plants and growth mediums were measured and compared between exposed plants and control plants. These exposure experiments have explored the effects and relevant mechanisms of NO2 exposure on uptake and metabolism of nitrate and ammonium, on translocation and distribution of N within plant organs, on activity of N2-fixation, as well as the effects of N supplies to roots on NO2 absorption by leaves. In addition, two new calculation methods were proposed to improve the estimation methods for NO2 absorption by plants. Exposing soybean plants, supplied with nitrate, to high levels of NO2 (1.1 µl 1-1) caused accumulations of ammonium and nitrate in leaves, decreases in nitrate uptake by roots and in the amount of organic N per plant, and increases in the acidities of both leaves and nutrient solution. These results suggest that an increase in H+ from NO2 absorption in exposed plants may inhibit ammonium assimilation, and convert HCO3- to H2CO3 in roots, causing the decrease in nitrate uptake by roots. The H+ from NO2 absorption may be neutralized by the OH- produced from nitrate reduction, or it may exude into the growth medium through roots. If the H+ cannot be promptly removed from exposed plants, the H+ accumulation may inhibit general assimilation and uptake of nitrate. NO2 absorption, as a supplement to N supply, can partly meet the N demand of plants; and the N derived from NO2 absorbed by leaves was mainly located in leaves. Consequently NO2 exposure of plants supplied with nitrate usually decreases the nitrate transport from roots to leaves and the redistribution of N within plants. Exposing the soybean plants, grown with ammonium, to 0.2 - 0.25 µl 1-1 of NO2 increased ammonium concentration in leaves, slowed down the rise in leaf acidity from ammonium assimilation, and decreased ammonium uptake by roots. These results suggest that reduction of the nitrate and nitrite, produced from N02 absorption, consumes H+ and competes for energy with ammonium assimilation which produces H+. It inhibits the increase in concentration of H+ from ammonium assimilation in exposed plants. As plants take up ammonium through exchange of the H+ in roots with the ammonium in nutrient solution, the lower H+ concentration in exposed plants decreases ammonium uptake by roots, compared with control plants. This inhibition of ammonium uptake may further restrict ammonium assimilation and H+ increase in exposed plants. Long-term exposure of soybean plants to 0.2 µl 1-1 of NO2 increased concentrations of nitrite and H+ in root nodules, and inhibited activity of nitrogenase. Nitrate supply to roots intensified the nitrite accumulation, decreased the acidity in nodules, and alleviated the inhibition of nitrogenase activity. These results suggest that the H+ from N02 absorption accelerates reduction of nitrite to NO and conversion of LHb to LHbNO, resulting in inhibition of nitrogenase activity. Reduction of nitrate in plants consumes H+, which may be the reason why nitrate supply can alleviate the inhibitory effects of NO2 on nitrogenase activity. Leaf pH and absorption rate of NO2 of the plants growing in a nitrate solution were higher than those of the plants growing in ammonium. The absorption rates of NO2 of both sets of plants decreased with increasing the duration of NO2 exposure. These results suggest that accumulation of H+ in leaves, caused by NO2 absorption and ammonium assimilation, may slow down the dissolution of NO2 in extracellular fluid of leaves, and result in the decrease in absorption rate of NO2. The decline in NO2 absorption with increasing exposure time, which is analogous to a decline in exposure concentration of NO2, may be one of the reasons why plants show some acclimation to long-term exposure. The popular methods for quantitative estimation of NOx absorption by leaves of plants supplied with 15N-labelled fertilizer, the 15N dilution method and the difference method, were compared with the classical mass balance (MB) method and/or the 15NOx method. Neglect of the influence of NOx exposure on N uptake by roots results in an overestimation in NO2 absorption calculated using the 15N dilution method. Two new alternative calculation methods, the TNF method and the TWF method, are proposed on different prerequisites. The TNF method can reduce the error of 15N dilution method, and give closer results to the MB method or 15NOx method. The TWF method is suitable to calculate NOx-N in organs (leaf, stem, root) of exposed plants. The estimation error of these methods are found to increase with decreasing concentration of 15N-labelled fertilizer supplied to roots. The results of these exposure experiments suggest that the acid-alkali balance plays a fundamental role in determining the interactions between NO2 absorption and N metabolism in exposed plants. H+ not only take part in the reactions of uptake and assimilation of various forms of nitrogen, it also affects the activity of the enzymes catalyzing reduction of oxidized N and synthesis of organic N. NO2 and other forms of N interact on each other through production or consumption of H+ in their uptake and assimilation. Whether the concentration of the H+ produced from NO2 absorption and N assimilation in exposed plants can be kept at a suitably low level, may determine whether the effects of NOx exposure on plants are beneficial or detrimental. According to the role of H+ in uptake and metabolism of N, two conclusions are possible: 1. Susceptibility of plants to NO2 exposure may depend on the acidity of the growth medium. 2. Velocities of uptake and assimilation of nitrogen in plants may fluctuate cyclically. Further experiments to test these inferences are recommended.

The abi3-1 mutant causes moderate perturbation of seed metabolism relative to wild-type seeds, so offering a distinct and discrete treatment for use in experiments investigating regulation of allocation between sources and sinks. abi3-1 plants continue to initiate new flowers, and hence siliques, for longer than wild-type plants. Total rates of carbon assimilation in the short-term were the same in both genotypes during early reproductive growth. This rate fell to 50-70 % in wild-type plants during later reproductive growth, but did not change in the mutant, consistent with delayed senescence of cauline leaves in abi3-1 plants. It was found that restriction of carbon and/or nitrogen availability restricted growth in both genotypes, and abolished the mutant phenotype. Specific leaf areas increased under shading and decreased when nitrate was limiting. Reduction in nitrate limitation from 90 to 80 % was considerably less limiting for wild-type plant growth, but remained grossly limiting for abi3-1 plants. In previously non-acclimatised plants of both genotypes, no difference was found in assimilation and allocation of ¹⁴C-radiolabel supplied at 200 ppm CO 2. 800 ppm CO 2 similarly had no effect on the wild-type, but caused abolition or inversion of normal source-sink relationships in abi3-1 plants. A significantly large amount of radiolabel was initially incorporated into starch in all tissues in abi3-1 plants, and later moved into the water soluble fraction in each tissue, most likely as sucrose. It is proposed that resource allocation is regulated by competition for resources between sinks maintaining sucrose concentration gradients in the phloem, and that sucrose is both the transport and signalling molecule in the mechanism described. No difference in concentration of sucrose in tissues was found between genotypes, but it was found that mutant siliques imported [U-¹⁴C]glucose into siliques significantly more slowly from the phloem than wild-type siliques. In conclusion, abi3-1 seeds may be seen as having reduced capacity for growth which causes stimulation of floral meristem development by feedback of sucrose in the phloem. Silique initiation is thereby prolonged, creating a demand for assimilates that delays cauline leaf senescence.

[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.

Orientador: Paulo Arruda
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O amino ácido lisina é catabolizado em plantas e animais pela via da sacaropina. Nesta via, a lisina é convertida a ?-aminoadipato-?-semialdeído (AASA) pela ação da enzima bifuncional lisina-cetoglutarato redutase/sacaropina desidrogenase (LKR/SDH). O intermediário AASA é então convertido a ?-aminoadipato (AAA) pela enzima ?-aminoadipato-?-semialdeído desidrogenase (AASADH). A LKR/SDH já foi bem caracterizada em plantas e animais, mas a atividade enzimática bem como o possível papel fiiológico da AASADH ainda não foi demonstrada em plantas. A via da sacaropina, além do seu importante papel na regulação dos níveis de lisina, está também envolvida em processos de resposta a estresses. Este trabalho está dividido em dois capítulos. No capítulo I descrevemos a identificação do gene que codifica a AASADH em milho e a caracterização da atividade enzimática da enzima em endosperma imaturo de milho. Mostramos que a AASADH é codificada pelo gene Aldh7b1, um gene muito conservado em eucariotos. A enzima codificada pelo gene Aldh7b1 foi parcialmente purificada de endosperma imaturo de milho e através de eletroforese em condições denaturantes e cromatografia em coluna de gel filtração mostramos que a enzima, na sua forma nativa, apresenta-se como um tetrâmero constituído por quatro subunidades de 55 kDa. A AASADH isolada de endosperma imaturo de milho converte o semi-aldeido AASA em AAA. O produto da reação catalisada pela AASADH foi confirmado por cromatografia em camada delgada. No capítulo II discutimos o papel da via sacaropina no desenvolvimento da semente e na resposta de planas jovens de milho a estresses abióticos. As enzimas LKR/SDH e AASADH são co-expressos nas células das camadas da sub-aleurona do endosperma de milho nas fases intermediarias do desenvolvimento. No entanto, embora a proteína AASADH seja produzida no endosperma e no embrião de sementes imaturas e nos tecidos de plantas jovens, a proteína LKR/SDH é detectada unicamente nas células da sub-aleurona das sementes imaturas. A AASADH mostrou atividade máxima a pH 7,4 e Kms para AASA e NAD+ na ordem de micromolar. Em endosperma imaturo a via da sacaropina é induzida por lisina e reprimida por estresse salino, enquanto prolina e ácido pipecólico são significativamente reprimidos por lisina. Em coleóptiles jovens as enzimas LKR/SDH e AASADH são induzidas transcricionalmente por estresses salino, osmótico e oxidativo, mas enquanto que a proteína AASADH acumula nos tecidos sob estresse, a proteína LKR/SDH não é detectada. Nossos resultados indicam que os genes que codificam as enzimas LKR/SDH e AASADH são co-expressos a nível transcricional, mas não a nível traducional. A ausência da proteína LKR/SDH em plantas jovens sob estresses e os altos níveis do seu transcrito serem detectados mostra um desacoplamento transcrição/tradução que podem ter consequências regulatórias ainda desconhecidas
Abstract: Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into ?-aminoadipate-?-semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into ?-aminoadipate (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). LKR/SDH was characterized in higher eukaryotes, but AASADH has not been demonstrated in plants. Furthermore, studies have shown that besides the degradation of lysine, the saccharopine pathway is involved in stress response processes in plants, animals and bacteria. This work was divided into two chapters. Chapter I describes the identification of the gene encoding AASADH and the partial purification and characterization of the enzyme from developing maize endosperm. The enzyme AASADH is encoded by the Aldh7b1 gene, a gene highly conserved among eukaryotes. The enzyme partially purified from developing endosperm and analyzed by SDS-PAGE and gel filtration chromatography behaved, in its native form, as a tetramer constituted by four monomers of 55 kDa. The enzymatic convertion of AASA into AAA was verified by thin layer chromatography. In Chapter II the role of the saccharopine pathway in seed development and stress responses is discussed. LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing seeds. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD+ in the micromolar range. In the developing endosperm the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in stressed tissues, LKR/SDH does not. Our results indicate that the genes encoding the LKR/SDH and AASADH enzymes are co-expressed at the transcriptional level, but not the translational level. The absence of LKR/SDH protein in young plants under stress despite of the high levels of transcripts being detected suggests a decoupling transcription/translation that may have regulatory consequences yet unknown
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of host nutrient dependency, allowing comparisons that provide insight into parasite adaptation. A key aspect of this is the relationship between parasite metabolism and the metabolite profile of its host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor. First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts that had mutations in amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. This research highlights the importance of aspartate and asparagine to early post-attachment metabolism in both P. aegyptiaca and T. versicolor and through host deficiencies possibly associated with decreased growth in P. aegyptiaca. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.
Ph. D.
Parasitic weeds of the plant family Orobanchaceae attach to the roots of host plants via haustoria. Parasite haustoria embed into the host plant and are capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of the extent to which a parasitic plant may depend on its host for nutrients. This allows comparisons that provide insight into the ways in which parasites adapt. A key aspect of this is the relationship between the metabolite profile of the parasite and the metabolite profile of the host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate parasite Phelipanche aegyptiaca (which cannot perform photosynthesis) and the facultative parasite Triphysaria versicolor (which can perform photosynthesis). First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts. These hosts had mutations in enzymes related to amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.

Orientador: Sonia Nair Bao
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A família Amaranthaceae no sensu lato, incluindo Chenopodiaceae, é formada por cerca de 2.360 espécies, 145 delas encontradas no Brasil; 94 espécies subsistem em diversas fitofisionomias do Bioma Cerrado, 71 são endêmicas de diferentes regiões e biomas brasileiros e 27 aparecem em listas regionais de espécies ameaçadas de extinção. Visando contribuir para o conhecimento das espécies dessa família, foram estudados aspectos ecológicos, micromorfológicos, anatômicos e ultraestruturais de folhas de Amaranthaceae nativas do Brasil, com ênfase nos gêneros Alternanthera, Gomphrena, Froelichiella, Hebanthe e Pfaffia. As espécies nativas de regiões abertas de cerrado apresentam adaptações que favorecem a sobrevivência em condições adversas (seca e fogo), tais como raízes tuberosas ou lenhosas e xilopódios, hábito herbáceo e/ou subarbustivo, pilosidade densa nas porções aéreas, senescência de ramos aéreos durante as fases mais secas, dependência de fogo ou chuva para rebrotação e/ou floração, frutificação rápida seguida de dispersão anemocórica, epidermes com cutículas bem desenvolvidas e metabolismo fotossintético C4. O comportamento pirofítico das espécies favorece o estabelecimento pioneiro das mesmas, principalmente nas áreas abertas do Cerrado. As superfícies foliares de algumas espécies do gênero Gomphrena apresentam cristais de cera epicuticular do tipo plaquetas, orientadas paralelamente, aspecto anteriormente descrito apenas em espécies de Chenopodiaceae. Em duas espécies foram encontrados fungos Ascomyceto colonizando folhas, cujos aspectos ultraestruturais foram descritos. A anatomia Kranz foi caracterizada em seis espécies do gênero Gomphrena, que também possuem cloroplastos dimórficos, demonstrando estrutura foliar compatível com o metabolismo fotossintético C4. Na análise das duas espécies do gênero Alternanthera (uma C3 e outra intermediária C3-C4) verificou-se que a posição das organelas nas células da bainha pode ser um elemento chave na determinação do tipo metabólico. As espécies de Froelichiella, Hebanthe e Pfaffia possuem anatomia e ultraestrutura compatíveis com o metabolismo C3. A anatomia foliar e a ultraestrutura das espécies estudadas apresentam um padrão já descrito para outras espécies da família Amaranthaceae, exceto para os gêneros Hebanthe e Froelichiella, cuja descrição anatômica e ultraestrutural foi realizada pela primeira vez. A evolução do metabolismo C4 pode estar relacionada, pelo menos em parte, ao desenvolvimento da anfiestomia associada à maior espessura do limbo foliar em espécies herbáceas. Plastoglóbulos bem desenvolvido foram encontrados em cloroplastos de algumas das espécies do Cerrado e parecem associados aos mecanismos de defesa, além do metabolismo de lipídios. A família Amaranthaceae pode ser um bom marcador da biodiversidade de dicotiledôneas de pequeno porte e da capacidade de regeneração das áreas de campos rupestres, campos úmidos e outros tipos de vegetação aberta dos cerrados. A riqueza de informações obtidas durante o estudo de espécies dessa família ilustram a importância da ampliação das pesquisas básicas e aplicadas em suas espécies, especialmente as que ocorrem naturalmente no Cerrado. A ampliação do conhecimento relativo à anatomia e à especialização das organelas de diferentes gêneros para o metabolismo fotossintético pode contribuir para o entendimento da evolução da via C4 e do ambiente onde as plantas se especializaram. O estudo do Bioma Cerrado pretende ampliar as justificativas para esforços de preservação de sua biodiversidade.
Abstract: The Amaranthaceae family sensu lato, including Chenopodiaceae, is comprised of approximately 2,360 species, 145 of them found in Brazil. 94 species exist in various vegetation types of the Cerrado, 71 are endemic to different regions and Brazilian biomes, and 27 appear in regional lists of endangered species. To contribute to the knowledge of this family species the ecological aspects, micromorphology, anatomy and ultrastructure of leaves of brazilian Amaranthaceae were studied, with emphasis on the genera Alternanthera, Gomphrena, Froelichiella, Hebanthe and Pfaffia. The native species of open areas of Cerrado (a savannah-like vegetation) exhibit adaptations that promote survival in adverse conditions (drought and fire), such as tuberous or woody roots, xylopodium, herbaceous or subshrub habit, dense pubescence in aerial portions, senescence of shoots and leaves during the driest season, dependence on rain or fire to resprout and/or flowering, fruiting followed by rapid wind dispersion, tick cuticle on epidermis and C4 photosynthetic metabolism. The species' fire behavior favors their establishment as pioneers, especially in open areas of the Cerrado. Leaf surfaces of some species of the genus Gomphrena present epicuticular wax crystals in platelet form, oriented in parallel, an aspect previously described only in Chenopodiaceae species. In two of the species studied Ascomycete fungi were found colonizing its leaves, and ultrastructural aspects were described. Kranz anatomy was found in six species of the genus Gomphrena, which also have dimorphic chloroplasts, showing leaf structure compatible with the C4 photosynthetic metabolism. In the analysis of two species of Alternanthera (a C3 and a C3-C4 intermediate) showed that besides the Kranz anatomy, the position of organelles in bundle sheath cells can be a key element in determining the metabolic type. The Froelichiella, Hebanthe and Pfaffia species have leaf anatomy and ultrastructure consistent with C3 metabolism. The leaf anatomy and ultrastructure have a pattern already described for other species of the Amaranthaceae family, except for the genera Hebanthe and Froelichiella, whose anatomical and ultrastructural aspects were described for the first time. The evolution of C4 metabolism may be related, at least in part, to the development of amphystomy associated with increased leaf thickness in herbaceous species. Large plastoglobuli were found in chloroplasts of some Cerrado species and appear associated with defense mechanisms, and lipid metabolism. The Amaranthaceae family can be a good marker of the biodiversity of nonwoody Eudicotyledons with ability to regenerate areas of rocky fields, wet grasslands and other open vegetation of the cerrado. These results illustrate the importance of expansion of basic and applied research in Amaranthaceae species, especially those that occur naturally in Brazilian cerrados. The expansion of knowledge concerning the anatomy and the specialization of organelles of different genera to perform its photosynthetic metabolism may contribute to understanding of the evolution of C4 pathway and the environment where plants specialize. The study of Cerrado Biome aims to broaden the justification for efforts to preserve its biodiversity.
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

The endoplasmic reticulum (ER) stress response is an important signal transduction pathway that senses ER stress caused by misfolded proteins or increased secretory protein traffic and induces molecular chaperone expression to counter such stress. The response has been well characterized in yeast and mammals where it has been associated with a variety of metabolic pathways, such as phospholipid biosynthesis, translational inhibition, and ER associated degradation. In plants, however, the connections of the ER stress response with metabolic pathways other than those involved in chaperone biosynthesis have not been characterized. This study defines a connection between phospholipid synthesis and the ER stress response in plants. Two model systems were used to characterize this association, the maize mutant floury-2 (fl2), which displays a unique endosperm specific ER stress response mediated by a mutant seed storage protein, and soybean cell cultures treated with the pharmacological agents tunicamycin (Tm) or azetidine-2-carboxylic acid (AZC). These chemicals interfere with normal protein synthesis and processing events, and are well characterized inducers of ER stress responses in animals, plants, and yeast. Investigation of both of these systems revealed a common theme; induction of the ER stress response in plants leads to increased activity and/or expression of various phospholipid biosynthetic enzymes. These increases were correlated with previously described amplifications in expression of the molecular chaperones binding protein (BiP), protein disulfide isomerase (PDI) and calreticulin. Certain aspects of the ER stress response may be unique to plants. A seed-specific result of the ER stress response was the accumulation of triacylglycerols (TG), which specifically increased in the endosperm of the fl2 mutant to more than 3-fold higher than normal endosperm levels by 36 days after pollination (DAP). The maize and soybean systems used in this study provide a starting point for the investigation of other details of the ER stress response in plants and represent important tools for future efforts to define the components of the signaling pathway.

Plants produce a variety of small molecules, including those essential for survival in all conditions (primary metabolites) or for more ecologically specific conditions (secondary metabolites). While primary metabolic pathways are broadly shared among plants, secondary metabolism is under constant selective pressure towards chemical innovation, given the continual fluctuation of the environment. Thus, plant secondary metabolism - whose constituents number in the hundreds of thousands - is lineage-specific, highly structurally diverse, and ultimately of high value to medicine, agriculture, and industry. Efforts to optimize the production of specific metabolites or to discover new compounds remain difficult primarily due to inadequate understandings of the metabolic genes involved and how these genes are regulated. This work first examines co-regulation, a major form of organization by which plant secondary metabolic genes are organized. In response to the bacterial crop pathogen Pseudomonas syringae, Arabidopsis thaliana and its relatives in the mustard family produce numerous secondary metabolites from the amino acid tryptophan, including the antimicrobial compound camalexin. However, hundreds of biosynthetic genes of unknown function are also simultaneously upregulated. Using metabolic profiling and co-expression analysis, I helped to identify the complete biosynthetic pathway to the indole-3-carbonylnitriles (ICNs), a previously unknown class of compounds. When the cytochrome P450 gene CYP82C2 is mutated, biosynthesis of the compound 4-hydroxy-ICN (4OH-ICN) is abolished, and plant defense against P. syringae is impaired. Conversely, addition of 4OH-ICN to plants is sufficient to suppress bacterial growth. Next, this work examines the evolution of camalexin and 4OH-ICN metabolism. Cytochrome P450-directed secondary metabolism has been shown almost without exception to be evolutionarily derived from changes to enzymes with broad substrate specificity. By contrast, I observe through genetics, enzyme phylogenetic analysis, and transient expression assays that the ICN and camalexin biosynthetic pathways evolved from a common chemical substrate. In particular, changes to camalexin catalysis by the newly duplicated gene CYP71A12 led to the formation of ICN metabolism in several mustard species, although both compounds are directly derived from indole cyanohydrin. Furthermore, 40H-ICN is an extremely recently evolved metabolite, derived from a flurry of genic, epigenetic and transposon-mediated rearrangements of a yet-more recent gene duplicate (CYP82C2). These regulatory changes to CYP82C2 lead to its pathogen-inducibility solely in the species A. thaliana. I additionally identify WRKY33 and MYB51 as two sets of defense regulators that carefully fine-tune 40H-ICN metabolism by direct biosynthetic gene regulation. WRKY33 transcription factor, which is involved in the species-specific regulation of CYP82C2, is conserved throughout flowering plants, indicating that transcriptional recruitment is an important feature in the expansion of secondary metabolism. Finally, this work probes possible molecular functions of 40H-ICN and camalexin by exploring the molecular mechanisms underlying their secretion from roots and regulation of cell death processes. This study ultimately reveals that the proliferation of diverse chemical arsenals in plants is greatly aided by the regulatory capture of new and rapidly evolving genes by evolutionarily more stable transcription factors. Future emphases on transcriptional regulators of secondary metabolism may thus aid in the discovery of new secondary metabolic pathways on a more rapid scale.

The effect of boron deficiency was studied in tomato plants grown hydroponically. Morphological studies showed that boron deficiency in the root system caused a cessation of root elongation, browning and thickening of root tips, the formation of branch roots close to the apex and decrease in the overall size of the root. The pyrimidine biosynthetic pathway remained intact and functional under boron limitation. The sugar nucleotide, uridine diphosphate glucose, was diminished in boron deficient medium and its level was restored to that of born sufficiency when uracil, cytosine, uridine, cytidine or orotic acid was provided.

Sustainable and environmentally friendly agriculture represents the main strategy to meet rising food needs and to contribute to a stable planet. To achieve these goals, one of the most promising solutions is the adoption of plant biostimulants. Biostimulants are natural compounds which, at low concentrations, are able to enhance plant nutrient uptake, nutrient efficiency, tolerance to abiotic/biotic stresses, and crop yield. Humic substances (HS) fall within the biostimulants definition, as they have positive effects on the uptake of macro and micronutrients that considerably improve the metabolism, the growth and yields of relevant agricultural crops. With this background, the aims of this Ph.D. project were to i) evaluate metabolic and biochemical status of plants treated with HS to elucidate the action mechanisms of plant biostimulants. This might permit the development of a second generation of biostimulants where synergies and complementary mechanism can be functionally designed. ii) investigate potential biostimulant effects of a fungicide compound. In manuscript I Arabidopsis thaliana plants were treated with HS for eight hours. Root proteins were studied by MS spectrometry coupled with iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. 902 protein spots were identified for HS treated roots, where 291 proteins were differentially expressed. Bioinformatic tools such as DAVID, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of the network amongst the identified proteins. From this analysis, it was possible to evaluate that most of the differentially expressed proteins were functionally classified into response to inorganic substances, redox homeostasis, energy metabolism, protein synthesis, cell trafficking and division. With the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Moreover, from the analysis of interactomes and DAVID clusters it was possible to observe previously undiscovered HS effects, i.e. on the Ubiquitin and RACK1A interactome subnetworks. In manuscript II Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analysed by 1H NMR an FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrates contents, and leaf amino acids from protein hydrolysis were identified and quantify by gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC/MS) techniques. Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters. EF treatment had the highest influence on metabolites profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants resulted not significant different from the control. A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity. In manuscript III the biostimulant activity of HS extracted from four leonardites is analysed on maize seedlings. After 48 h of treatment with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L−1) of HS, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analyzed. HS from different leonardites had similar spectroscopic pattern, with small differences. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols. This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. In the manuscript IV it was investigated the potential biostimulant side-effects of sedaxane. Physiological changes in disease-free maize seedlings at increasing application doses (25, 75 and 150 μg a.i. seed-1) under controlled sterilised conditions were analysed. Sedaxane had significant auxin-like and gibberellin-like effects, with maximum benefits attained at the intermediate dose. Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids). It is concluded that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages. As a general conclusion, even if HS were extracted from different sources were applied on different plant species, they displayed similar biological activities. Proteomics and metabolomics studies confirmed that “–omics” techniques are essential tools to have a ‘panoramic’ view on metabolic changes happening inside an organism after a positive or negative external perturbation.
L'agricoltura conservativa rappresenta la strategia principale per soddisfare le crescenti esigenze alimentari e contribuire a un pianeta stabile. Per raggiungere questi obiettivi, una delle soluzioni più promettenti è l'adozione dei biostimolanti. I biostimolanti sono composti naturali che, a basse concentrazioni, sono in grado di migliorare l'assorbimento dei nutrienti delle piante, l'efficienza dell’attività dei nutrienti, la tolleranza agli stress abiotici / biotici e la resa delle colture. Le sostanze umiche (HS) sono considerate una classe di biostimolanti, in quanto hanno effetti positivi sull'assorbimento di macro e micronutrienti, migliorano considerevolmente il metabolismo, la crescita e le rese delle colture agricole. Con questo background, gli obiettivi di questo progetto di dottorato sono stati di i) valutare lo stato metabolico e biochimico delle piante trattate con HS per chiarire i meccanismi di azione dei biostimolanti. Ciò potrebbe consentire lo sviluppo di una seconda generazione di biostimolanti in cui meccanismi di azione sinergici e complementari possono essere considerati. ii) studiare potenziali effetti biostimolanti di un composto fungicida. Nel primo articolo le piante di Arabidopsis thaliana sono state trattate con HS per otto ore. Le proteine delle radici sono state studiate mediante spettrometria MS accoppiata con la tecnica iTRAQ (Isobaric Tag for Relative and Absolute Quantification). Per le radici trattate con HS sono stati identificate 902 proteine, dove 291 proteine erano espresse in modo differenziale. Strumenti bioinformatici come DAVID, KEGG, IIS e Cytoscape sono stati utilizzati per interpretare le funzioni biologiche, l'analisi e la visualizzazione delle proteine connesse identificate. Da questa analisi, è stato possibile valutare che la maggior parte delle proteine differenzialmente espresse sono state funzionalmente classificate in risposta alle sostanze inorganiche, redox omeostasi, metabolismo energetico, sintesi proteica, traffico e divisione cellulare. Con il presente studio è stata fornita una panoramica sulle vie metaboliche più influenzate dall'attività biologica delle HS. Inoltre, dall'analisi degli interattomi e dei cluster di DAVID è stato possibile osservare nuovi effetti di HS, come per esempio sugli interattomi dell’Ubiquitina e del RACK1A. Nel secondo articolo, le piantine di Arabidopsis thaliana, coltivate in condizioni idroponiche, sono state trattate per 8 ore con acido indol-3-acetico (IAA), HS da International Humic Substances Society (IHSS) e HS da feci di lombrico (EF), rispettivamente. Le caratteristiche strutturali delle sostanze umiche sono state analizzate mediante spettroscopie 1H NMR e FT-IR. Nelle foglie e nelle radici, mediante gascromatografia-spettrometria di massa (GC / MS) e tecniche di cromatografia liquida-spettrometria di massa (LC / MS) sono stati identificati e quantificati gli amminoacidi, zuccheri e il contenuto di carboidrati liberi, e gli amminoacidi fogliari ottenuti dall'idrolisi proteica. L'analisi discriminante canonica (CDA) è stata utilizzata per valutare l'influenza dei trattamenti sui parametri studiati. Il trattamento con EF ha avuto la massima influenza sui profili dei metaboliti rispetto al controllo, IAA e IHSS. L'analisi del CDA ha evidenziato una chiara distinzione tra le risposte fisiologiche delle piante trattate con EF e IHSS, in base alle diverse proprietà chimiche e strutturali delle HS. Le piante trattate con IAA non sono invece risultate significativamente differenti dal controllo. Una migliore comprensione degli effetti specifici delle diverse HS, anche in relazione alle loro caratteristiche chimiche, potrebbe servire come base per l'identificazione di composti marcatori per la bioattività delle HS. Nel terzo articolo, l'attività biostimolante delle HS estratte da quattro leonarditi è stata analizzata su piantine di mais. Dopo 48 ore di trattamento con 5 concentrazioni (0, 0,1, 0,5, 1 e 10 mg CL-1) di HS, sono stati analizzati la crescita e la morfologia delle radici, l’attività della glutammina sintetasi (GS), l’attività del glutammato sintasi (GOGAT), il contenuto proteico totale, il contenuto di zuccheri solubili, l'attività della fenilalanina ammonia-liasi (PAL), i fenoli solubili e gli acidi fenolici liberi. Le HS estratte da diverse leonarditi avevano un pattern spettroscopico simile, ma con piccole differenze. HS_USA ha indotto una maggior crescita delle radici, della superficie radicale e della proliferazione delle radici secondarie. HS_4, HS_USA e HS_B hanno determinato una miglior efficienza dell'uso dei nutrienti delle piante, incrementando l'attività degli enzimi GS e GOGAT e la produzione totale di proteine. Le quattro HS hanno stimolato anche l'attività dell'enzima PAL, seguita da una maggiore produzione di fenoli solubili totali. Questo studio ha rilevato che, sebbene l'attività delle HS dipendesse dall'origine della leonardite, questi composti possono essere attribuiti ai prodotti biostimolanti, poiché stimolano la crescita delle piante, il metabolismo dell'azoto e l'accumulo di sostanze fenoliche. Nel quarto articolo è stato studiata l’azione biostimolante del sedaxane, intesa come attività secondaria. Sono stati analizzati i cambiamenti fisiologici in piantine di mais all'aumentare delle dosi di applicazione (25, 75 e 150 μg a.i. seme-1) in condizioni sterili controllate (piante non infette). Sedaxane ha dimostrato avere effetti di tipo auxino- e gibberellino-simili significativi, con i massimi benefici raggiunti alla dose intermedia. La lunghezza della radice (+ 60% rispetto ai controlli non trattati), l'area (+ 45%) e le biforcazioni (+ 51%) sono stati significativamente influenzati. Sedaxane ha incrementato l'attività della glutammina sintetasi (GS) delle foglie e delle radici con conseguente maggiore accumulo di proteine. Sedaxane ha anche incrementato l'attività della fenilalanina ammonia-liasi (PAL) nelle foglie, che potrebbe essere responsabile dell'aumento dell'attività antiossidante (acidi fenolici). Si è concluso che, oltre al suo effetto protettivo, il sedaxane può facilitare lo sviluppo delle radici e intensificare il metabolismo dell'azoto e dei fenilpropanoidi nelle giovani piante di mais e può essere utile per superare gli stress biotici e abiotici nelle prime fasi di crescita. Come conclusione generale, anche se le HS sono state estratte da diverse matrici e applicate su diverse specie di piante, queste hanno mostrato attività biologiche simili. Studi di proteomica e metabolomica hanno confermato che le tecniche "-omics" sono strumenti essenziali per avere una visione "panoramica" sui cambiamenti metabolici che avvengono all'interno di un organismo dopo una perturbazione esterna positiva o negativa.

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.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O pinhão-manso (Jatropha curcas L.) é uma planta conhecida e cultivada na América desde tempos remotos, porém, somente nas últimas três décadas passou a ser estudado agronomicamente. É considerada uma espécie promissora no processo de produção de biocombustíveis devido ao seu elevado teor de óleo nas sementes. Por tratar-se de uma planta ainda em fase de domesticação, os estudos acerca de seu crescimento e fisiologia ainda são escassos, porém necessários para a expansão da cultura em todo o país, por isso, o objetivo desta pesquisa foi analisar o crescimento e o metabolismo do nitrogênio de plantas jovens de J. curcas cultivadas sob quatro condições de sombreamento (0, 30, 50 e 70 % de cobertura) e quatro concentrações de nitrato (0, 15, 30 e 45 mM). Durante 120 dias as plantas foram tratadas e avaliadas biometricamente quanto à altura, diâmetro, número de folhas, área foliar e massas fresca e seca. Fisiologicamente analisou-se a atividade das enzimas redutase do nitrato e glutamina sintetase, proteína total solúvel, análise quantitativa de nitrato e amônia. Os resultados obtidos sugerem que com relação ao crescimento inicial J. curcas é responsiva ao sombreamento, como características ecofisiológicas que se refletem em adaptações morfológicas de expansão da área foliar, alongamento do pecíolo e maior crescimento em altura buscando ambientes com maior luminosidade, sendo que ambientes com sombreamento de 30% são eficazes no controle da temperatura foliar e retenção de água no substrato permitindo o melhor desenvolvimento das plantas, entretanto, níveis acima de 50% de sombreamento são prejudiciais ao crescimento inicial da espécie por provocarem intenso estiolamento e redução no número médio de folhas. A realização da adubação nitrogenada é de suma importância...
The jatropha (Jatropha curcas L.) is a plant known in America and cultivated since ancient times, but only in the last three decades began to be studied agronomy. It is considered a promising species for the biofuels production because its high oil content in seeds. It‟s a plant still under domestication because it, studies on their growth and physiology are still scarce, but necessary to the expansion of culture throughout the country, so the objective of this research was to analyze the growth and nitrogen metabolism in young plants of J. curcas grown under four conditions of shading (0, 30, 50 and 70% coverage) and four nitrate concentrations (0, 15, 30 and 45 mM). For 120 days the plants were treated and biometrically evaluated as to height, diameter, number of leaves, leaf surface area and fresh and dry mass. Physiologically, we analyzed the enzymes activity of the nitrate reductase and glutamine synthetase, total soluble protein, quantitative analysis of nitrate and ammonia. The results suggest that with respect to the initial growth J. curcas is responsive to the shading, with physiologic characteristics that reflected in morphological adaptations of leaf area expansion, petiole elongation and greater height growth looking environments with higher brightness, and shading environments with 30% are effective in leaf temperature control and water retention in the substrate allowing for better development of plants, however, levels above 50% shading is detrimental to early growth of the species to cause intense etiolation and reduction in the number of leaves. The nitrogen fertilizer is a paramount importance in the early development of plants, the start deficiency visual symptoms appear in the first month after germination. Nitrate concentrations exceeding 30 mM are harmful because they cause a reduction... (Complete abstract click electronic access below)

CAPES
Durante a dormência, as plantas de clima temperado cessam temporariamente o crescimento visível. No caso da macieira sabe-se que a temperatura é o fator ambiental que mais influencia a dormência. A planta necessita de baixas temperaturas para induzir a entrada e posteriormente a saída da dormência, para então retomar o crescimento vegetativo. Sabe-se que quando não ocorre acúmulo suficiente de frio, as plantas apresentam crescimento errático, com brotação ocorrendo predominantemente na porção distal dos ramos, comprometendo a brotação satisfatória. No entanto, há pouca informação sobre o metabolismo destes carboidratos e sua relação com a dormência de plantas em condições de inverno ameno. Contudo o objetivo deste trabalho foi avaliar as diferenças biológicas e bioquímicas entre as porções distal e proximal de ramos de macieira que influenciam a acrotonia. De abril a outubro dos anos de 2012, 2013 e 2014, foram coletados ramos e esporões de três cultivares de macieira (Eva, Galaxy e Fuji Suprema) cultivadas em um pomar comercial no município de Plamas-PR. Para avaliar a intensidade da dormência em gemas vegetativas foi realizado o teste de estacas de uma só gema, e o teste de Tabuenca para as gemas florais. Variáveis bioquímicas também foram avaliadas como o teor de proteínas solúveis; atividade de alfa-amilase; açúcares (glicose, frutose, sacarose e sorbitol); amido e umidade. De forma geral as cultivares apresentaram tempo médio de brotação (TMB) menores do que em clima temperado. A porcentagem de brotação manteve-se alta na maioria das coletas para as três cultivares. O teste de Tabuenca confirmou a precocidade da cultivar Eva e mostrou comportamento muito semelhante entre a ‘Galaxy’ e a ‘Fuji Suprema’. Além disso foi observado um intervalo muito curto entre a saída da endodormência em condições de forçagem e em condição de campo. A umidade ponderal variou conforme o padrão da temperatura em cada ano, sendo que em 2013, ano de maior acúmulo de frio a umidade ponderal apresentou os menores valores para todas as cultivares. Em 2013 a tendência de aumento da atividade da alfa-amilase e no teor de proteína coincidiram com o menor valor de TMB. A glicose e frutose apresentaram comportamento muito semelhantes entre si durante as observações. O teor de sacarose variou de ano para ano, não apresentando um padrão fixo. O sorbitol, açúcar mais abundante no xilema, tende a ser mais constante durante a dormência com leve tendência de redução próximo à brotação. O teor de amido tende a diminuir com os primeiros acúmulos de horas frio, no entanto frequentemente sofre ressíntese, devido às ondas de calor comuns no inverno. Existe diferenças entre as porções dital e proximal dos ramos para todas as variáveis analisadas, com teor de carboidrato, atividade enzimática, proteínas e umidade predominantemente maiores na porção distal dos ramos, com algumas exceções pontuais. Isso pode estar associado à ocorrência de acrotonia em macieiras. As plantas avaliadas parecem não entrar em dormência profunda em condição de inverno ameno, tendo seu metabolismo muito sujeito às oscilações de temperatura. O metabolismo das plantas tende a mudar de ano para ano de acordo com o padrão do inverno em cada ano.
During dormancy, temperate plants temporarily stops visible growth. In apple trees, it is known that temperature is the environmental factor that most influences dormancy. The plant requires low temperatures to induce input and then to output endodormancy, thus resumption vegetative grow. It is known that when there isn’t sufficient chilling accumulation, plants present erratic growth, with budbreak occurring predominantly in the distal portion of the branches, compromising the satisfactory budbreak. However, there is little information on the metabolism of these carbohydrates and their relationship to plant dormancy in subtropical humid climates. This work aimed to evaluate the biological and biochemical differences between the distal and proximal portions of apple tree branches that influence acrotonia. From April to October of 2012, 2013 and 2014, branches and spurs of three apple cultivars (Eva, Galaxy and Fuji Suprema) were harvested in a commercial orchard in Palmas-PR. In order to evaluate the dormancy intensity in vegetative and floral buds, single bud cuttings test and Tabuenca test were carried out, respectively. Biochemical variables were also evaluated such as soluble protein content; Alpha-amylase activity; Soluble sugars (glucose, fructose, sucrose and sorbitol); Starch and water content. In general, the cultivars showed the Mean Time to Budbrst (MTB) lower than in temperate climate. The budburst percentage remained high in most of the observation dates for the three cultivars. The Tabuenca test confirmed the precocity of the cultivar Eva and showed very similar behavior between 'Galaxy' and 'Fuji Suprema'. In addition, a very short interval between the endodormancy output under forcing conditions and in field condition was observed. The water content varied according to the temperature pattern in each year, and in 2013, the year of greatest cold accumulation, the lowest values for all cultivars were observed. In 2013, the trend of increased alpha-amylase activity and protein content coincided with the lower value of MTB. Glucose and fructose presented very similar behavior among themselves during the observations. The sucrose content varied from year to year and did not present a fixed pattern. Sorbitol, sugar most abundant in the xylem, tends to be more constant during dormancy with a slight reduction tendency close to budding. The starch content tends to decrease with the first chilling accumulations, however it often undergoes resynthesis due to the heat waves common in winter. There are differences between the distal and proximal portions of the branches for all variables analyzed, with carbohydrate content, enzymatic activity, proteins and humidity predominantly larger in the distal portion of the branches, with some occasional exceptions. This may be associated with the occurrence of acrotonia in apple trees. The evaluated apple trees do not appear to fall into deep endodormancy in a mild winter condition, their metabolism seems to be very subject to temperature oscillations. Plant metabolism tends to change from year to year according to the winter pattern in each year.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches.
AFRIKAANSE OPSOMMING: Ondanks verskeie pogings, waartydens verskeie gene geteiken is, is daar nog weinig sukses behaal om sukrose-akkumulering te verhoog. Toenemend wil dit voorkom asof suksesvolle genetiese manipulering van sukroseberging in die stingel van die verandering van ‘n nuwe geen afhanklik sal wees. Een van die moontlike benaderings wat gevolg kan word om potensiële teiken gene wat ‘n belangrike rol in die beheer van sukrose-opberging speel te identifiseer, is om geen uitdrukkingspatrone in die stingel tydens die omskakeling van aktiewe groei tot volwassenheid te karakteriseer. In hierdie studie is ‘n metode gebaseer op die hibridisering van mRNA geoptimiseer en suksesvol aangewend om die uitdrukkingspatrone van 90 verskillende geselekteerde gene, wat vir sleutelensieme in die beheer van koolhidraatmetabolisme kodeer, te bestudeer. Die doel met die ondersoek was om die beperkte kennis oor die regulering van koolhidraatmetabolisme uit te brei en om die bestaande inligting afgelei van fisiologiese en biochemiese-studies aan te vul. Drie verskillende mRNApopulasies, verkry uit verskillende dele van die stingel, is ontleed deur verskillende peilers te gebruik. Die gegewens is statisties ontleed en dit het afleidings oor die verandering in uitdrukking van hierdie gene moontlik gemaak. Die relatiewe konsentrasies van 74 verskillende gene is gedokumenteer. Gene wat sterk uitgedruk word het aldolase, heksokinase, sellulase, alkoholdehidrogenase en ongebonde suurinvertase ingesluit. Die uitdrukkingspatrone van 15 gene het tussen die verkillende weefsels gevarieer. Gene waarvan die uitdrukking tydens die oorgang na volwassenheid verlaag sluit in UDP-glukose pirofosforilase en UDP-glukose dehidrogenase en waarvan die uitdrukking verhoog sukrosefosfaatsintase, sukrosesintase en neutrale invertase in. Die uitdrukking van verskeie suikertransporter gene verhoog tydens volwassewording. Hierdie inligting te same met die huidige kennis oor heersende metabolietvlakke sal bydrae tot die identifisering van geenteikens vir toekomstige genetiese manupulering.

Orientador: Enes Furlani Júnior
Banca: Liliane Santos de Camargos
Banca: Enio Tiago de Oliveira
Resumo: O pinhão-manso (Jatropha curcas L.) é uma planta conhecida e cultivada na América desde tempos remotos, porém, somente nas últimas três décadas passou a ser estudado agronomicamente. É considerada uma espécie promissora no processo de produção de biocombustíveis devido ao seu elevado teor de óleo nas sementes. Por tratar-se de uma planta ainda em fase de domesticação, os estudos acerca de seu crescimento e fisiologia ainda são escassos, porém necessários para a expansão da cultura em todo o país, por isso, o objetivo desta pesquisa foi analisar o crescimento e o metabolismo do nitrogênio de plantas jovens de J. curcas cultivadas sob quatro condições de sombreamento (0, 30, 50 e 70 % de cobertura) e quatro concentrações de nitrato (0, 15, 30 e 45 mM). Durante 120 dias as plantas foram tratadas e avaliadas biometricamente quanto à altura, diâmetro, número de folhas, área foliar e massas fresca e seca. Fisiologicamente analisou-se a atividade das enzimas redutase do nitrato e glutamina sintetase, proteína total solúvel, análise quantitativa de nitrato e amônia. Os resultados obtidos sugerem que com relação ao crescimento inicial J. curcas é responsiva ao sombreamento, como características ecofisiológicas que se refletem em adaptações morfológicas de expansão da área foliar, alongamento do pecíolo e maior crescimento em altura buscando ambientes com maior luminosidade, sendo que ambientes com sombreamento de 30% são eficazes no controle da temperatura foliar e retenção de água no substrato permitindo o melhor desenvolvimento das plantas, entretanto, níveis acima de 50% de sombreamento são prejudiciais ao crescimento inicial da espécie por provocarem intenso estiolamento e redução no número médio de folhas. A realização da adubação nitrogenada é de suma importância... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The jatropha (Jatropha curcas L.) is a plant known in America and cultivated since ancient times, but only in the last three decades began to be studied agronomy. It is considered a promising species for the biofuels production because its high oil content in seeds. It‟s a plant still under domestication because it, studies on their growth and physiology are still scarce, but necessary to the expansion of culture throughout the country, so the objective of this research was to analyze the growth and nitrogen metabolism in young plants of J. curcas grown under four conditions of shading (0, 30, 50 and 70% coverage) and four nitrate concentrations (0, 15, 30 and 45 mM). For 120 days the plants were treated and biometrically evaluated as to height, diameter, number of leaves, leaf surface area and fresh and dry mass. Physiologically, we analyzed the enzymes activity of the nitrate reductase and glutamine synthetase, total soluble protein, quantitative analysis of nitrate and ammonia. The results suggest that with respect to the initial growth J. curcas is responsive to the shading, with physiologic characteristics that reflected in morphological adaptations of leaf area expansion, petiole elongation and greater height growth looking environments with higher brightness, and shading environments with 30% are effective in leaf temperature control and water retention in the substrate allowing for better development of plants, however, levels above 50% shading is detrimental to early growth of the species to cause intense etiolation and reduction in the number of leaves. The nitrogen fertilizer is a paramount importance in the early development of plants, the start deficiency visual symptoms appear in the first month after germination. Nitrate concentrations exceeding 30 mM are harmful because they cause a reduction... (Complete abstract click electronic access below)

Polyribosome aggregation state in growing tissues of barley and wheat leaf or stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf or stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of ³²PO₄³⁻ into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) ⁺RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) ⁺RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system; activity of nonpolysomal mRNP was variable, but usually less than that of polysomal mRNP. Deproteinization of mRNP failed to improve its activity. No inhibition of translation of poly(A) ⁺RNA by nonpolysomal mRNP was observed in mixing experiments with the wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described.

Transketolase is a TPP dependent enzyme that affects the availability of intermediates in both the Calvin cycle and non-oxidative pentose phosphate pathway. Previous studies have indicated that changes to the activity level of transketolase can limit growth and development as well as the production of isoprenoids, starch, amino acids and thiamine. The overall aim of this project was to further advance the understanding of the mechanism linking increased TK activity and thiamine metabolism. Nicotiana tabacum mutants with increased total transketolase activity ~ 2 to 2.5 fold higher than WT plants were shown to have a reduced growth and chlorotic phenotype. In seedlings, these phenotypes were attributed to a reduction in seed thiamine content. Imbibition of TKox seeds in a thiamine solution produced plants that were comparable to WT plants. However, the chlorotic but not growth phenotype was found to return unless the plants underwent irrigation with a thiamine solution indicating that TKox plants are unable to produce sufficient quantities of thiamine to meet demand. Furthermore, the application of deoxy-xylulose-5-phosphate was also found to be able to partially complement the phenotype suggesting that flux from the C3 cycle into the non-mevalonate pathway is being affected. Analysis of thiamine and TPP levels demonstrated that TKox plants were deficient in thiamine but not TPP in the majority of cases. In plants that had begun to flower, TKox lines had reduced thiamine and TPP levels in the 20th fully open leaf compared to the same leaf in WT plants. Furthermore, sampling of leaf tissue from both WT and TKox seedlings at the same developmental stage indicated that high levels of TK protein may lead to the accumulation of TPP in these areas causing a reduction in the levels of thiamine and TPP in the rest of the plant thereby limiting growth and development.

Els processos metabòlics en plantes, com ara la fotosíntesi, la fotorespiració i la respiració, comporten l’inevitable producció d’espècies reactives de l’oxigen (ROS) en cloroplasts, peroxisomes i mitocondris. En determinades concentracions les ROS poden actuar com a molècules implicades en la senyalització cel•lular, però degut a la seva elevada reactivitat, un augment de les ROS provoca l’oxidació de components cel•lulars alterant-ne la seva funció biològica i provocant dany oxidatiu a la planta. En condicions d’estrès, tant biòtic com abiòtic, les plantes experimenten un increment dels nivells de ROS. Per tal de mantenir l’homeòstasi redox, aquestes han desenvolupat un seguit de mecanismes antioxidants capaços de reduir els nivells de ROS evitant un possible dany oxidatiu. La recerca per desxifrar les respostes de les plantes a l’estrès ha anat en augment en els últims anys, però encara avui en dia és poc el coneixement que tenim sobre els mecanismes implicats en el cas de les plantes perennes, tot i que constitueixen una part molt important del regne vegetal. Per altra banda, tot i l’evident importància de l’estrès oxidatiu, són escassos els estudis que s’han plantejat com aquest es pot veure afectat per factors intrínsecs de la planta, com l’edat o el sexe. L’objectiu principal d’aquesta tesi ha estat determinar com l’edat de la planta, tant la cronològica com la fisiològica, i el sexe poden influir en l’estrès oxidatiu en plantes perennes. L’anàlisi del nivells d’estrès oxidatiu es va dur a terme mitjançant les mesures de diferents mecanismes antioxidants com els carotenoides, l’alfa-tocoferol i els antocians, però centrant-nos sobretot en el paper de l’àcid malondialdehid, un subproducte de la peroxidació lipídica. Els estudis es van realitzar en fulles i plantes juvenils de Pistacia lentiscus L., una espècie dioica i perenne típica del clima mediterrani, i en arbres moribunds de Fagus sylvatica L., proporcionant un bon model a causa de la seva avançada edat, tant cronològica com fisiològica. Els resultats obtinguts revelen que l’augment dels nivells de peroxidació lipídica com a indicador d’estrès oxidatiu pot significar un dany o un possible mecanisme de senyalització interna; per això, la consideració conjunta de l’edat cronològica i els nivells d’estrès oxidatiu és un bon indicador de l’edat fisiològica, tant a nivell de fulla com de planta sencera. L’esforç reproductiu en plantes dioiques ocasiona canvis en els mecanismes fotoprotectors en les femelles respecte als mascles en condicions ambientals adverses. Tot i que les femelles presenten uns nivells d’estrès oxidatiu superiors als dels mascles, no es veuen afectades negativament, el que suggereix un possible rol en senyalització. Així mateix, l’estudi a nivell modular mitjançant la comparació entre brots reproductius i no reproductius en femelles va revelar una major fotoprotecció en els brots reproductius, com indicaven els nivells d’antioxidants i la major dissipació d’excés d’energia en forma de calor, emfatitzant la importància de la diferenciació entre mòduls en l’estudi de les diferències entre sexes en plantes dioiques.
Metabolic processes in plants such as photosynthesis, photorespiration and respiration, produce reactive oxygen species (ROS). ROS are highly toxic molecules but besides of its damaging nature they are implicated in cell signaling in different cellular processes. However, under stress conditions plants can suffer an increase of ROS levels. When ROS concentration becomes high enough to overwhelm antioxidant systems, plant suffer oxidative stress as a consequence of the unbalanced cellular redox status. Despite the importance to unravel plant stress responses, little is known about the mechanisms implicated in perennial plants. Furthermore, the possible effect of plant intrinsic factors, as plant age or reproductive effort, in oxidative stress levels is still poorly understood. The main objective of this thesis was to determine how plant age, both chronological and physiological, as well as reproductive effort may influence oxidative stress levels in perennial plants. With this purpose levels of antioxidants as carotenoids, anthocyanins and alpha-tocopherol together with endogenous contents of stress hormones were measured, but with special emphasis in malondialdehyde acid levels, a byproduct of lipid peroxidation. To better understand the plant age effect we used leaves and juvenile plants of Pistacia lentiscus as well as moribund beech trees. Increases in lipid peroxidation not only could mean an oxidative damage but play a signaling role. Therefore, chronolorgical age concomitantly with the measure of oxidative stress levels is a good indicator of plant physiological age. Sex-related changes in photoprotection mechanisms between female and male plants of Pistacia lentiscus, a dioecious plant, where observed under climatological adverse conditions. Although females phowed higher oxidative stress levels compared to males, females were not affected negatively, suggesting a role in signaling. In addition, photoprotection capacity was higher in reproductive shoots relative to non-reproductive shoots in females, thus suggesting that females prioritized protection to fruit-bearing shoots.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Most terrestrial plants make use of beneficial symbiotic associations to obtain nutrients (eg. nitrogen (N) and phosphorous (P)) from fungi in exchange for photoautotrophic carbon. However, plant parasitism (defined here as the ability of certain plants to parasitize other living material) has evolved in the plant kingdom and such plants obtain some, or all, of their nutritional needs from a host, which is severely negatively impacted by the parasite. While the physiological adaptations are well studied, the underlying molecular and biochemical mechanisms of plant parasitism remain largely unknown. As a first approach, a biochemical blueprint of primary metabolites present within parasitic plant species was constructed. The metabolomes of nineteen parasitic plants, ranging from hemi- and holoparasitism to mycoheterotrophism, were profiled via gas chromatography mass spectrometry (GC MS) based technology and targeted spectrophotometric assays. Based on these analyses, three important observations were made. First, parasitic plants were severely carbon deprived, despite being successful in colonizing and exploiting their hosts. Second, the levels of organic acids participating in mitochondrial respiration decreased and certain amino acids and soluble protein content increased. This suggests that parasitic plants utilize alternative respiratory substrates to compensate for a limitation in carbon supply. Third, although characterized by reduced carbohydrate pools, minor sugars normally not associated with plant metabolism, dominated the soluble sugar pool. The presence and significance of one of these sugars, namely turanose (α-D-glucopyranosyl-(1→3)-α-D-fructofuranose), was further investigated. Turanose biosynthetic reactions could be demonstrated in Orobanche minor extracts. Protein purification and mass spectrometry identification suggested that turanose biosynthesis occurred uniquely in parasitic plants. Future work will elucidate the functional significance of turanose metabolism in plant parasitism. Taken together, this study significantly contributes to our understanding of plant parasitism through development of metabolic signatures associated with distinct parasitic classes. These biochemical profiles highlighted several important strategies and alternative metabolic pathways that are either expressed or constitutively activated during parasitism. This knowledge broadens the scope of using parasitic plants in several biotechnological applications or as a novel research tool to address fundamental questions in plant science.
AFRIKAANSE OPSOMMING: Meeste landelike plante maak gebruik van voordelige simbiotiese assosiasies met swamme om voedinsgtowwe (bv. stikstof (N) en fosfor (P)) van hulle te verkry in ruil vir koolstof geproduseer deur die plant. Plant parasitisme (gedefinieer hier as die vermoë van sekere plante om ander lewende materiaal te parasiteer) het ontwikkel in die planteryk waar hulle sommige, of al hul voedings stowwe van 'n gasheer plant ontvang, wat erg negatief geraak word deur die parasiet. Terwyl die fisiologiese aanpassings goed gebestudeer is, is die onderliggende molekulêre en biochemiese meganismes van plant parasitisme steeds grootliks onbekend. As 'n eerste benadering, was hierdie projek geïnisieer om 'n biochemiese bloudruk op te bou van primêre metaboliete teenwoordig in parasitiese plante. Die metabolome van negentien parasitiese spesies, wat wissel van hemi - en holoparasiete tot mikoheterotrofiese plante, is ondersoek deur gas chromatografie – massa spektrometrie (GC MS) gebaseerde tegnologie en geteikende spektrofotometriese toetse. Gebaseer op hierdie ontledings was drie belangrike waarnemings gemaak. Eerstens, parasitiese plante was erg koolstof arm, ten spyte daarvan dat hulle suksesvol is in die aanhegting en ontginning van voedingstowwe vanaf gasheer plante. Tweedens, die vlakke van organiese sure wat deelneem aan mitochondriale respirasie het afgeneem, terwyl sekere aminosure en oplosbare proteïen inhoude toegeneem het. Dit dui daarop dat parasitiese plante gebruik maak van alternatiewe respiratoriese substrate om te vergoed vir 'n beperking in koolstof aanbod. Derde, alhoewel parasitiese plante gekenmerk word deur verminderde koolhidraat inhoude, het skaarse suikers wat normaalweg nie verband hou met plant metabolisme nie, hulle oplosbare suiker inhoud oorheers. Die teenwoordigheid en betekenis van een van hierdie suikers, naamlik turanose (α -D -glucopyranosyl-(1→3)-α-D-fructofuranose), was verder ondersoek. Die sintese reaksie van turanose kan gedemonstreer word in Orobanche hederae uittreksels. Proteïen suiwering en massa spektrometrie identifikasie het voorgestel dat turanose biosintese uniek plaasvind in parasitiese plante. Toekomstige werk sal aandui wat die betekenis is van turanose metabolisme in plant parasitisme. Saamgevat het hierdie studie aansienlik bygedra tot ons begrip van plant parasitisme deur ontwikkeling van metaboliese handtekeninge wat verband hou met onderskeie parasitiese klasse. Hierdie biochemiese profiele beklemtoon verskeie belangrike strategieë en alternatiewe metaboliese paaie wat óf uitgedruk of konstitutief geaktiveer word tydens parasitisme. Hierdie kennis verbreed die omvang van die gebruik van parasitiese plante in verskeie biotegnologiese toepassings of as 'n nuwe navorsings instrument om fundamentele vrae in plant wetenskap aan te spreek.