Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.

Expression of the mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) in tobacco leaves leads to phosphate export.

Phosphate homeostasis in multicellular eukaryotes depends on both phosphate influx and efflux. The mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) shares homology to the Arabidopsis PHO1, a phosphate exporter expressed in roots. However, phosphate export activity of XPR1 has not yet been demonstrated in a heterologous system. Here, wedemonstrate that transient expression in tobacco leaves of XPR1-GFP leads to specific phosphate export. Like PHO1-GFP, XPR1-GFP is localized predominantly to the endomembrane system in tobacco cells. These results show that tobacco leaves are a good heterologous system to study the transport activity of members of the PHO1/XPR1 family.

Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers.

In shade-intolerant plants such as Arabidopsis, a reduction in the red/far-red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non-DNA-binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G-boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non-DNA-binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.

How do plants feel the heat?

In plants, the heat stress response (HSR) is highly conserved and involves multiple pathways, regulatory networks and cellular compartments. At least four putative sensors have recently been proposed to trigger the HSR. They include a plasma membrane channel that initiates an inward calcium flux, a histone sensor in the nucleus, and two unfolded protein sensors in the endoplasmic reticulum and the cytosol. Each of these putative sensors is thought to activate a similar set of HSR genes leading to enhanced thermotolerance, but the relationship between the different pathways and their hierarchical order is unclear. In this review, we explore the possible involvement of different thermosensors in the plant response to warming and heat stress.

Cuticular defects lead to full immunity to a major plant pathogen.

In addition to its role as a barrier, the cuticle is also a source of signals perceived by invading fungi. Cuticular breakdown products have been shown previously to be potent inducers of cutinase or developmental processes in fungal pathogens. Here the question was addressed as to whether plants themselves can perceive modifications of the cuticle. This was studied using Arabidopsis thaliana plants with altered cuticular structure. The expression of a cell wall-targeted fungal cutinase in A. thaliana was found to provide total immunity to Botrytis cinerea. The response observed in such cutinase-expressing plants is independent of signal transduction pathways involving salicylic acid, ethylene or jasmonic acid. It is accompanied by the release of a fungitoxic activity and increased expression of members of the lipid transfer protein, peroxidase and protein inhibitor gene families that provide resistance when overexpressed in wild-type plants. The same experiments were made in the bodyguard (bdg) mutant of A. thaliana. This mutant exhibits cuticular defects and remained free of symptoms after inoculation with B. cinerea. The expression of resistance was accompanied by the release of a fungitoxic activity and increased expression of the same genes as observed in cutinase-expressing plants. Structural defects of the cuticle can thus be converted into an effective multi-factorial defence, and reveal a hitherto hidden aspect of the innate immune response of plants.

The endodermis.

A Casparian strip-bearing endodermis is a feature that has been invariably present in the roots of ferns and angiosperms for approximately 400 million years. As the innermost cortical layer that surrounds the central vasculature of roots, the endodermis acts as a barrier to the free diffusion of solutes from the soil into the stele. Based on an enormous body of anatomical and physiological work, the protective endodermal diffusion barrier is thought to be of major importance for many aspects of root biology, reaching from efficient water and nutrient transport to defense against soil-borne pathogens. Until recently, however, we were ignorant about the genes and mechanisms that drive the differentiation of this intricately structured barrier. Recent work in Arabidopsis has now identified the first major players in Casparian strip formation. A mechanistic understanding of endodermal differentiation will finally allow us to specifically interfere with endodermal barrier function and study the effects on plant growth and survival under various stress conditions. Here, I critically review the major findings and models related to endodermal structure and function from other plant species and assess them in light of recent molecular data from Arabidopsis, pointing out where the older, descriptive work can provide a framework and inspiration for further molecular dissection.

Genome-wide transcriptional responses to shade: Linking shade avoidance and auxin

Plants have the ability to use the composition of incident light as a cue to adapt development and growth to their environment. Arabidopsis thaliana as well as many crops are best adapted to sunny habitats. When subjected to shade, these plants exhibit a variety of physiological responses collectively called shade avoidance syndrome (SAS). It includes increased growth of hypocotyl and petioles, decreased growth rate of cotyledons and reduced branching and crop yield. These responses are mainly mediated by phytochrome photoreceptors, which exist either in an active, far-red light (FR) absorbing or an inactive, red light (R) absorbing isoform. In direct sunlight, the R to FR light (R/FR) ratio is high and converts the phytochromes into their physiologically active state. The phytochromes interact with downstream transcription factors such as PHYTOCHROME INTERACTING FACTOR (PIF), which are subsequently degraded. Light filtered through a canopy is strongly depleted in R, which result in a low R/FR ratio and renders the phytochromes inactive. Protein levels of downstream transcription factors are stabilized, which initiates the expression of shade-induced genes such as HFR1, PIL1 or ATHB-2. In my thesis, I investigated transcriptional responses mediated by the SAS in whole Arabidopsis seedlings. Using microarray and chromatin immunoprecipitation data, we identified genome-wide PIF4 and PIF5 dependent shade regulated gene as well as putative direct target genes of PIF5. This revealed evidence for a direct regulatory link between phytochrome signaling and the growth promoting phytohormone auxin (IAA) at the level of biosynthesis, transport and signaling. Subsequently, it was shown, that free-IAA levels are upregulated in response to shade. It is assumed that shade-induced auxin production takes predominantly place in cotyledons of seedlings. This implies, that IAA is subsequently transported basipetally to the hypocotyl and enhances elongation growth. The importance of auxin transport for growth responses has been established by chemical and genetic approaches. To gain a better understanding of spatio-temporal transcriptional regulation of shade-induce auxin, I generated in a second project, an organ specific high throughput data focusing on cotyledon and hypocotyl of young Arabidopsis seedlings. Interestingly, both organs show an opposite growth regulation by shade. I first investigated the spatio-transcriptional regulation of auxin re- sponsive gene, in order to determine how broad gene expression pattern can be explained by the hypothesized movement of auxin from cotyledons to hypocotyls in shade. The analysis suggests, that several genes are indeed regulated according to our prediction and others are regulated in a more complex manner. In addition, analysis of gene families of auxin biosynthetic and transport components, lead to the identification of essential family members for shade-induced growth re- sponses, which were subsequently experimentally confirmed. Finally, the analysis of expression pattern identified several candidate genes, which possibly explain aspects of the opposite growth response of the different organs.

Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching.

Protophloem is a specialized vascular tissue in growing plant organs, such as root meristems. In Arabidopsis mutants with impaired primary root protophloem differentiation, brevis radix (brx) and octopus (ops), meristematic activity and consequently overall root growth are strongly reduced. Second site mutation in the protophloem-specific presumed phosphoinositide 5-phosphatase COTYLEDON VASCULAR PATTERN 2 (CVP2), but not in its homolog CVP2-LIKE 1 (CVL1), partially rescues brx defects. Consistent with this finding, CVP2 hyperactivity in a wild-type background recreates a brx phenotype. Paradoxically, however, while cvp2 or cvl1 single mutants display no apparent root defects, the root phenotype of cvp2 cvl1 double mutants is similar to brx or ops, although, as expected, cvp2 cvl1 seedlings contain more phosphatidylinositol-4,5-biphosphate. Thus, tightly balanced phosphatidylinositol-4,5-biphosphate levels appear essential for proper protophloem differentiation. Genetically, OPS acts downstream of phosphatidylinositol-4,5-biphosphate levels, as cvp2 mutation cannot rescue ops defects, whereas increased OPS dose rescues cvp2 cvl1 defects. Finally, all three mutants display higher density and accelerated emergence of lateral roots, which correlates with increased auxin response in the root differentiation zone. This phenotype is also created by application of peptides that suppress protophloem differentiation, CLAVATA3/EMBRYO SURROUNDING REGION 26 (CLE26) and CLE45. Thus, local changes in the primary root protophloem systemically shape overall root system architecture.

Female sterility associated with increased clonal propagation suggests a unique combination of androdioecy and asexual reproduction in populations of Cardamine amara (Brassicaceae).

BACKGROUND AND AIMS: The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites. METHODS: Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites. KEY RESULTS: Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %. CONCLUSIONS: The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.

Eficiência de transformação genética de citrange 'carrizo' com duas construções gênicas

Transformação genética é considerada uma importante ferramenta auxiliar no melhoramento genético de plantas cítricas. Entretanto, a eficiência de transformação pode variar em função de diversos fatores, incluindo a própria construção gênica utilizada. Este trabalho buscou avaliar a eficiência de transformação genética de plantas de citrange 'Carrizo' [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] com duas construções gênicas diferentes contendo o gene uidA (GUS) sob o controle dos promotores Arabidopsis thaliana phloem protein 2 (AtPhP2) e Arabidopsis thaliana sucrose transporter 2 (AtSuT2). Segmentos de epicótilo de plântulas germinadas in vitro foram utilizados como explantes. O gene nptII, que confere resistência ao antibiótico canamicina, foi utilizado nas construções gênicas como agente de seleção para regeneração de plantas transgênicas. O ensaio histoquímico com X-GLUC foi realizado em todas as brotações regeneradas para verificar a expressão do gene uidA. Dos 4.790 segmentos de epicótilo utilizados, registrou-se a regeneração de 366 brotações com reação positiva no ensaio histoquímico, as quais foram enxertadas em porta-enxertos cultivados in vitro. Cinco dessas brotações, de cada construção gênica, foram selecionadas para análise da PCR, com primers específicos para amplificação da sequência do gene uidA. A inserção do transgene foi confirmada por PCR em todas as brotações selecionadas. A eficiência de transformação e o número de brotos escapes, avaliada pelo teste histoquímico, variaram em função das construções gênicas utilizadas.

Trade-off between constitutive and inducible resistance against herbivores is only partially explained by gene expression and glucosinolate production.

The hypothesis that constitutive and inducible plant resistance against herbivores should trade-off because they use the same resources and impose costs to plant fitness has been postulated for a long time. Negative correlations between modes of deployment of resistance and defences have been observed across and within species in common garden experiments. It was therefore tested whether that pattern of resistance across genotypes follows a similar variation in patterns of gene expression and chemical defence production. Using the genetically tractable model Arabidopsis thaliana and different modes of induction, including the generalist herbivore Spodoptera littoralis, the specialist herbivore Pieris brassicae, and jasmonate application, constitutive and inducibility of resistance was measured across seven A. thaliana accessions that were previously selected based on constitutive levels of defence gene expression. According to theory, it was found that modes of resistance traded-off among accessions, particularly against S. littoralis, in which accessions investing in high constitutive resistance did not increase it substantially after attack and vice-versa. Accordingly, the average expression of eight genes involved in glucosinolate production negatively predicted larval growth across the seven accessions. Glucosinolate production and genes related to defence induction on healthy and herbivore-damaged plants were measured next. Surprisingly, only a partial correlation between glucosinolate production, gene expression, and the herbivore resistance results was found. These results suggest that the defence outcome of plants against herbivores goes beyond individual molecules or genes but stands on a complex network of interactions.

Multilayered organization of jasmonate signalling in the regulation of root growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Subcellular Redistribution of Root Aquaporins Induced by Hydrogen Peroxide.

Aquaporins are water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The present work focuses on the oxidative stress-induced redistribution of plasma membrane intrinsic protein (PIP) aquaporins from the plasma membrane (PM) to intracellular membranes. This process was investigated in the Arabidopsis root. Sucrose density gradient centrifugation showed that exposure of roots to 0.5 mM H2O2 induces significant depletion in PM fractions of several abundant PIP homologs after 15 min. Analyses by single-particle tracking and fluorescence correlative spectroscopy showed that, in the PM of epidermal cells, H2O2 treatment induces an increase in lateral motion and a reduction in the density of a fluorescently tagged form of the prototypal AtPIP2;1 isoform, respectively. Co-expression analyses of AtPIP2;1 with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in the late endosomal compartments. Life-time analyses established that the high stability of PIPs was maintained under oxidative stress conditions, suggesting that H2O2 triggers a mechanism for intracellular sequestration of PM aquaporins without further degradation. In addition to information on cellular regulation of aquaporins, this study provides novel and complementary insights into the dynamic remodeling of plant internal membranes during oxidative stress responses.

Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana. Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.