Spatially and genetically distinct control of seed germination by phytochromes A and B.

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

" Arabidopsis Thaliana " response to insect feeding : new components controlling defense gene expression and plant resistance

Abstract: Plants cannot run away to escape attacking herbivores, but they defend themselves by producing anti-digestive proteins and toxic compounds (for example glucosinolates). The first goal of this thesis was to study changes in gene expression after insect attack using microarrays. The responses of Arabidopsis thaliana to feeding by the specialist Pieris rapae and the generalist Spodoptera liffora is were compared. We found that the transcript profiles after feeding by the two chewing insects were remarkably similar, although the generalist induced a slightly stronger response. The second goal was to evaluate the implication of the four signals jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) in the control of insect-regulated gene expression. Using signaling mutants, we observed that JA was the predominant signal and that ABA modulated defense gene expression. In contrast, SA and ET appeared to control slightly gene expression, but only after feeding by S. litforalis. The third goal was to establish whether plant responses are really effective against insects. In accordance with the transcript profile, both insects were affected by the JA-dependent defenses, as they performed better on the JA-insensitive mutant. S. littoralis also performed better on ABA-deficient mutants, providing evidence for the role of ABA in defense against insects. When testing indole or aliphatic glucosinolate deficient mutants, we found that they were also more susceptible to insect feeding, providing some of the first genetic evidence for the defensive role of glucosinolates in planta. Finally, a glutathione-deficient mutant, pad2-1, was also more susceptible to insect feeding and we could attribute this phenotype to a lowered accumulation of the major indole glucosinolate. In this thesis, we provide a comprehensive list of insect-regulated genes, including many transcription factors that constitute interesting candidate genes for the further study of insect-induced expression changes. Understanding how the plant responses to insects are regulated will provide tools for a better management of insect pest in the field. Résumé: Les plantes ne peuvent s'échapper pour fuir les insectes qui les attaquent, mais elles se défendent en produisant des protéines anti-digestives et des composés toxiques (par exemple des glucosinolates). Le premier but de cette thèse était d'étudier les changements de l'expression génétique lors d'attaque par des insectes en utilisant des puces à ADN. Nous avons comparé la réponse d'Arabidopsis thaliana à deux espèces d'insectes avec des habitudes alimentaires différentes : le spécialiste Pieris rapae et le généraliste Spodoptera littoralis. Nous avons trouvé que les profils de transcription après l'attaque par les deux insectes sont remarquablement similaires, bien que le généraliste induise une réponse légèrement plus forte. Le deuxième but était de déterminer l'implication de quatre signaux dans le contrôle de la réponse :l'acide jasmonique (JA), l'acide salicylique (SA), l'éthylène (ET), et l'acide abscissique (ABA). En utilisant de mutants de signalisation, nous avons montré que l'acide jasmonique était le signal prédominant et que l'acide abscissique modulait l'expression génétique. D'autre part, l'acide salicylique et l'éthylène contrôlent à un degré moindre l'expression génétique, mais seulement après l'attaque par S. littoralís. Le troisième but était d'établir si les réponses des plantes sont efficaces contre les insectes. En accord avec le profil de transcription, les deux espèces d'insectes se sont mieux développées sur un mutant insensible au JA, indiquant que les défenses contrôlées par ce signal sont cruciales pour la plante. De plus, les larves de S. littorales se sont mieux développées sur des mutants déficients en ABA, ce qui fournit une preuve du rôle de l'acide abscissique dans la défense contre les insectes. En testant des mutants déficients en glucosinolates de type indole ou aliphatique, nous avons trouvé qu'ils étaient plus sensibles aux insectes, démontrant ainsi le rôle défensif des glucosinolates in planta. Finalement, le mutant déficient en glutathion pad2-1 était aussi plus sensible à l'attaque des insectes, et nous avons pu attribuer ce phénotype à une plus faible augmentation d'un indole glucosinolate dans ce mutant. Dans cette thèse, nous avons mis en évidence un nombre important de gènes contrôlés par les insectes, comprenant de nombreux facteurs de transcription qui constituent des candidats intéressants pour`étudier plus en détail les changements d'expression génétique induits par les insectes. Une meilleure compréhension de la réponse des plantes contre l'attaque des insectes devrait nous permettre de développer de nouvelles stratégies pour mieux gérer les ravageurs des cultures.

Polyamine metabolism and signaling in plant abiotic stress protection

Polyamines (PAs) are small polycationic compounds present in all living organisms. Compelling evidences indicate a role for PAs in plant protection against stress. During the recent years, genetic, molecular and ‘omic’ approaches have been undertaken to unravel the role of PAs in stress signaling. Overall, results point to intricate relationships between PAs, stress hormone pathways and ROS signaling. Such cross-regulations condition stress signaling through the modulation of abscisic acid (ABA) and ROS amplification-loops. In this chapter we compile our recent findings which elucidate molecular mechanisms and signalingpathways by which PAs contribute to stress protection in plants.

Signaling pathways controlling induced resistance to insect herbivores in Arabidopsis.

Insect attack triggers changes in transcript level in plants that are mediated predominantly by jasmonic acid (JA). The implication of ethylene (ET), salicylic acid (SA), and other signals in this response is less understood and was monitored with a microarray containing insect- and defense-regulated genes. Arabidopsis thaliana mutants coi1-1, ein2-1, and sid2-1 impaired in JA, ET, and SA signaling pathways were challenged with the specialist small cabbage white (Pieris rapae) and the generalist Egyptian cotton worm (Spodoptera littoralis). JA was shown to be a major signal controlling the upregulation of defense genes in response to either insect but was found to suppress changes in transcript level only in response to P. rapae. Larval growth was affected by the JA-dependent defenses, but S. littoralis gained much more weight on coi1-1 than P. rapae. ET and SA mutants had an altered transcript profile after S. littoralis herbivory but not after P. rapae herbivory. In contrast, both insects yielded similar transcript signatures in the abscisic acid (ABA)-biosynthetic mutants aba2-1 and aba3-1, and ABA controlled transcript levels both negatively and positively in insect-attacked plants. In accordance with the transcript signature, S. littoralis larvae performed better on aba2-1 mutants. This study reveals a new role for ABA in defense against insects in Arabidopsis and identifies some components important for plant resistance to herbivory.

Screening of Brazilian soybean genotypes with high potential for somatic embryogenesis and plant regeneration

The aim of this work was to identify Brazilian soybean (Glycine max) genotypes with potential to respond to in vitro culture stimuli for primary somatic embryo induction, secondary embryo proliferation and plant regeneration. Differences among eight tested cultivars were observed at each stage. Two cultivars, IAS-5 and BRSMG 68 Vencedora, were selected for the evaluation of the capacity for embryo differentiation and plant regeneration. These cultivars had high embryo induction frequencies, repetitive embryogenic proliferation, and low precocious embryo germination in the initial experiment. The effect of abscisic acid (ABA) and charcoal addition on plant regeneration was investigated. The addition of ABA to proliferation medium and of ABA and activated charcoal to maturation medium increased embryo differentiation rates, which resulted in a higher number of regenerated plants. The BRSMG 68 Vencedora cultivar was found to have a high potential for embryo induction, embryo proliferation and plant regeneration. The potential of this cultivar for somatic embryogenesis was similar to that observed for cultivar IAS-5, which is currently used for soybean transformation in Brazil. BRSMG 68 Vencedora may be a good alternative genotype for soybean genetic engineering via somatic embryogenesis protocols.

Physiological response of vicia faba to prohexadione-calcium under saline conditions

Changes in the activities of oxidative enzymes (indole acetic acid oxidase, peroxidase and catalase), endogenous hormones (gibberellic acid (GA3), indole acetic acid (IAA), abscisic acid (ABA) and cytokinins (AsZeatin), photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), total carbohydrates, total soluble sugars, amino acid proline and vegetative growth parameters were used as indicators to explain the physiological role of the growth retardant prohexadione-calcium on Vicia faba seedlings 40 days after sowing under salinity stress for 30 days. The obtained results show that soaking faba bean seeds prior to sowing at different concentrations of prohexadione-calcium (0, 10, 20 and 30 ppm) significantly increased the activities of indole acetic acid oxidase (IAA-oxidase) and peroxidase enzymes, but decreased the catalase enzyme activity as compared with their respective control. Application of prohexadione-Ca caused markedly decreases in the endogenous contents of gibberellins and indole acetic acid (IAA) but increased the levels of natural growth inhibitor abscisic acid (ABA) and cytokinins in the shoots of faba bean seedlings. All the prohexadione-Ca concentrations increased the contents of amino acid proline, photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), total carbohydrates and total soluble sugars in faba bean seedlings grown under salt stress. Application of prohexadione-Ca decreased significantly seedling height and shoot fresh weight but significantly increased shoot dry weight.

A model of seed dormancy in wild oats (Avena fatua) for investigating genotype x environment interactions

Dormancy is an adaptive trait in seed populations that helps ensure that seed germination is distributed over time and occurs in environmental conditions suitable for seedling growth. Several genes.. associated with seed dormancy in various plant species, have been integrated into a hypothetical dormancy model for Avena fatua L. (wild oats). Generally, the synthesis of, and sensitivity to, abscisic acid (ABA) during imbibition determines whether genes similar to those during maturation are expressed leading to a maintenance of dormancy during extended imbibition. Alternatively, there may be a shift towards expression of genes associated with gibberellins leading to germination. Environmental factors during maturation, after-ripening and imbibition are likely to interact with the genotype to affect gene expression and hence whether or not a seed germinates. In spite of the difficulties of working on a hexaploid species, A. fatua was selected for study because of its worldwide importance as a weed. Dormant and non-dormant genotypes of this species were also available. Gene expression studies are being carried out on three A.fatua genotypes produced tinder different environmental conditions to investigate the role of specific genes in dormancy and genotype X environment interactions in relation to dormancy.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage

Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacao L.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants.

In vitro micro-tuber initiation and dormancy in yam

Dormancy is a mechanism that regulates the timing of sprouting (germination) of affected plant parts as well as ensures that the food quality of edible parts is maintained in storage until the following growing season. In yam, however, little is known about the control of tuber initiation or tuber dormancy. The objective of this study was to determine the effects of selected plant growth regulators (PGRs) on tuber initiation and dormancy, using an in vitro system. In two replicated experiments, 2-chloroethylphosphonic acid (ethephon, an ethylene source), abscisic acid (ABA) and gibberellin (GA3) – and their inhibitors silver nitrate, fluridone and 2-chloroethyl-trimethylammonium chloride, respectively – were added at two concentrations to the culture medium prior to explant culture. Dates of micro-tuber initiation and sprouting (end of dormancy) and tuber number were recorded. In the control (no PGR) in Experiment 1, micro-tubers were initiated at the base of the stem after 176 days and sprouted 235 days later, that is 411 days after culturing. Most PGR treatments had only small effects (±30 days) on the duration of dormancy and the time of micro-tuber initiation. However, in GA3 micro-tuber initiation occurred after 76 days, about 100 days earlier than in the control, whereas fluridone affected the position of micro-tubers and duration of dormancy. With fluridone treatments, tubers were found at the base of the stem (normal position) and on lower and upper nodes. Lower node tubers sprouted within 225 days of culturing compared with about 420 days after culturing at other nodal positions and in other PGR treatments. These data suggest an important role for ABA and gibberellic acid in yam micro-tuber initiation and the induction of dormancy.

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

Overexpression and Purification of the Oat (Avena Fatua) Protein AFN1 and Construction of a pMAL/AFN1 Expression Plasmid

Abscisic acid (ABA) is an important phytohormone with regulatory roles in many physiological processes. ABA expression is induced by environmental stresses such as drought and it is known to be an inhibitor of seed germination. A wild oat (Avena fatua) called AFN1 has been hypothesized to initiate the early stages of germination as its mRNA accumulates in nondormant seed embryos during imbibition. The polypeptide sequence of AFN1 suggests that it is an ABA glucosyl transferase. Glucosylation by AFN1 and thereby inactivation of ABA could lead to seed germination. In order to understand the role of AFN1 in germination, an ample quantity of AFN1 polypeptide is needed to test for enzymatic ABA glucosylase activity. My work has been to overexpress recombinant AFN1containing a (His)6 tag using a pRSETC E.coli expression system followed by Purification of the AFN1 protein by means of a nickel-affinity column that bind to the (His)6 tag. Due to the insufficient yield of AFN1 fusion protein obtained with this procedure, another method using a pMAL-c2x vector is now being employed. The pMAL expression system provides a method for expressing and purifying protein by tagging proteins with maltose-binding protein (MBP). It is anticipated that MBP tag will be advantageous as it can make the fusion protein more soluble and thereby yield a larger quantity of protein. Currently, work is underway on the construction of pMAL/AFN1 plasmid.

Characterization of AFN1, a Gene Associated with Cereal Grain Germination

The AFN1 gene is transiently expressed in germinating oat grains. As AFN1 is not expressed in dormant oat grains during imbibition, we hypothesize that AFN1 may be involved in stimulating the germination process. Sequence analysis of an AFN1 cDNA clone indicates that the AFN1 polypeptide is similar to a previously identified abscisic acid (ABA) glucosyl transferase. This suggests that AFN1 may be acting to glucosylate ABA, thereby inactivating it. As the hormone ABA is known to inhibit germination, ABA glucosylation/inactivation could lead to germination in grains expressing AFN1. To test this hypothesis, we have constructed an expression plasmid that encodes an MBP::AFN1 (maltose binding protein) fusion protein. E. coli cells carrying the expression plasmid were found to produce the MBP::AFN1 fusion protein as a substantial fraction of total protein. We are currently in the process of purifying the MBP::AFN1 fusion protein by affinity chromatography, so that it can be assayed for ABA glucosyl transferase activity. We also wish to test the effect of AFN1 gene expression during grain imbibition on the germination behavior of the grains. To this end, we have constructed plasmids for the overexpression and RNAi-based suppression of AFN1 in transgenic plants. These plasmids have been introduced into oat cells by particle bombardment and we are in the process of regenerating transgenic plants for study.

Mechanism and control of Genipa americana seed germination

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Physiological and genomic variations in rice cells recovered from direct immersion and storage in liquid nitrogen

The use of cryoprotectants and slow cooling rates are routine procedures for the cryopreservation of plant cell lines. However, our results with rice (Oryza sativa L,, ev. Taipei 309) show that calli can be cryopreserved by direct immersion and stored in liquid nitrogen without any cryoprotection, the efficiency of recovery using this method, as well as a conventional method was generally increased with a previous abscisic acid (ABA) treatment. Following cryopreservation, calli demonstrated some differences with respect to unfrozen calli of the same lines, Thus, resistance to freezing stress (- 20 degrees C for 2 h) increased significantly in all lines tested, irrespective of their pre-incubation with ABA, Calli that had been directly stored in liquid nitrogen also demonstrated a higher competence for genetic transformation than their unfrozen counterparts, as indicated by the transient gene expression levels obtained after particle bombardment, These differences might lead to further biotechnological applications, A genetic analysis of amplified DNA polymorphisms was performed with three independent lines that had been subjected to four combinations of ABA treatment and direct immersion in liquid nitrogen, At the loci screened with the randomly amplified polymorphic DNA (RAPD) markers tested, the genetic variations among lines and among calli of the same line appear to bd more related to tissue-culture-induced somaclonal variation than to cryoselection.

Expressão gênica no embrião e no endosperma micropilar de sementes de café (coffea arabica L.) durante a germinação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)