MYB118 represses endosperm maturation in seeds of Arabidopsis.

In the exalbuminous species Arabidopsis thaliana, seed maturation is accompanied by the deposition of oil and storage proteins and the reduction of the endosperm to one cell layer. Here, we consider reserve partitioning between embryo and endosperm compartments. The pattern of deposition, final amount, and composition of these reserves differ between the two compartments, with the embryo representing the principal storage tissue in mature seeds. Complex regulatory mechanisms are known to prevent activation of maturation-related programs during embryo morphogenesis and, later, during vegetative growth. Here, we describe a regulator that represses the expression of maturation-related genes during maturation within the endosperm. MYB118 is transcriptionally induced in the maturing endosperm, and seeds of myb118 mutants exhibit an endosperm-specific derepression of maturation-related genes associated with a partial relocation of storage compounds from the embryo to the endosperm. Moreover, MYB118 activates endosperm-induced genes through the recognition of TAACGG elements. These results demonstrate that the differential partitioning of reserves between the embryo and endosperm in exalbuminous Arabidopsis seeds does not only result from developmental programs that establish the embryo as the preponderant tissue within seeds. This differential partitioning is also regulated by MYB118, which regulates the biosynthesis of reserves at the spatial level during maturation.

Mutations in the Arabidopsis homolog of LST8/GßL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days.

The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein ß subunit-like (LST8/GßL) protein is a member of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the synthesis of myo-inositol and raffinose during plant adaptation to long days.

Regulation of plant growth and metabolism by the TOR kinase.

The TOR (target of rapamycin) kinase is present in nearly all eukaryotic organisms and regulates a wealth of biological processes collectively contributing to cell growth. The genome of the model plant Arabidopsis contains a single TOR gene and two RAPTOR (regulatory associated protein of TOR)/KOG1 (Kontroller of growth 1) and GßL/LST8 (G-protein ß-subunit-like/lethal with Sec thirteen 8) genes but, in contrast with other organisms, plants appear to be resistant to rapamycin. Disruption of the RAPTOR1 and TOR genes in Arabidopsis results in an early arrest of embryo development. Plants that overexpress the TOR mRNA accumulate more leaf and root biomass, produce more seeds and are more resistant to stress. Conversely, the down-regulation of TOR by constitutive or inducible RNAi (RNA interference) leads to a reduced organ growth, to an early senescence and to severe transcriptomic and metabolic perturbations, including accumulation of sugars and amino acids. It thus seems that plant growth is correlated to the level of TOR expression. We have also investigated the effect of reduced TOR expression on tissue organization and cell division. We suggest that, like in other eukaryotes, the plant TOR kinase could be one of the main contributors to the link between environmental cues and growth processes.

Nitrogen signalling in Arabidopsis: how to obtain insights into a complex signalling network.

It is well known that nitrogen (N) and N status can be sensed by plants to regulate their development, physiology, and metabolism. Based on approaches efficiently used for fungi and algae, plant researchers have been trying, but with little success, to elucidate higher plants N signalling for several years. Recently, the use of new strategies such as transcriptomics, comparative reverse genetics, and new forward genetic screens have unravelled some players within the complex plant N signalling network. This review will mainly focus on these recent advances in the molecular knowledge of N sensing in plants such as the dual function of the nitrate transporter CHL1, the roles of the transcription factors LBD37/38/39 and NLP7 or of the CIPK8/23 kinases, as well as the implication of small RNAs, which are at last opening doors for future research in this field.

Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens.

WRKY genes encode proteins belonging to a large family of transcription factors that are involved in various developmental and physiological processes and in plant responses to pathogen infections. In the present work, a full-length cDNA from a Vitis vinifera L. cv. Cabernet Sauvignon grape berry library was isolated and characterized. The cDNA, designated VvWRKY2, encodes a polypeptide of 536 amino acids that shows the structural features of group I of WRKY protein family. VvWRKY2 is expressed in the different organs of healthy grapevine plants. In leaves, VvWRKY2 is induced by wounding and after infection with Plasmopara viticola. Constitutive expression of VvWRKY2 in tobacco reduced the susceptibility of transgenic tobacco to three types of fungal pathogens infecting different parts of the plant: Botrytis cinerea (leaves), Pythium spp. (roots) and Alternaria tenuis (seeds). The results indicate that VvWRKY2 may be involved in the resistance of grapevine against the pathogens.

Molecular basis of ergosterol-induced protection of grape against botrytis cinerea: induction of type I LTP promoter activity, WRKY, and stilbene synthase gene expression.

Type I lipid transfer proteins (LTPs) are basic, 9-kDa cystein-rich proteins believed to be involved in plant defense mechanisms. A 2,100-bp fragment containing the coding region of Vitis vinifera lipid transfer protein 1 (VvLTP1) and 1,420-bp of its promoter region was isolated by screening a grape genomic library. In silico analysis revealed several putative, defense-related, cis-regulatory elements such as W- and MYB-boxes, involved in the binding of WRKY and MYB transcription factors, respectively. The 5'-truncated versions of the VvLTP1 promoter were generated, cloned in front of the beta-glucuronidase (GUS) reporter gene, and introduced in tobacco plants and grapevine cell suspensions using Agrobacterium spp. Single MYB- and the W-boxes identified on the 0.250-kbp fragment were sufficient to induce GUS activity in transgenic tobacco plants after transient expression of MYB and WRKY. Ergosterol, a nonspecific fungal elicitor, induced GUS activity in transgenic grapevine cell suspensions transformed with the 1,420- and 750-bp promoter containing a palindromic arrangement of two W-boxes but not the 650- or 250-bp fragment, where only one W-box was present. Moreover, ergosterol triggered WRKY, VvLTP1, and stilbene synthase gene expression in grape plantlets and enhanced protection against Botrytis cinerea. The molecular basis of ergosterol-induced protection is discussed.

New insights on the organization and regulation of the fatty acid biosynthetic network in the model higher plant Arabidopsis thaliana.

In the plastids of plant cells, fatty acid (FA) production is a central biosynthetic process. It provides acyl chains for the formation of a variety of acyl lipids fulfilling different biological functions ranging from membrane synthesis to signaling or carbon and energy storage. The biochemical pathway leading to the synthesis of FA has been described for a long time. Over the last 15 years, and after the genome of the model higher plant Arabidopsis thaliana has been sequenced, the scientific community has deployed approaches of functional genomics to identify the actors comprising this pathway. One of the puzzling aspects of the emerging molecular biology of FA synthesis resided in the occurrence of multigene families encoding most enzymes of the pathway. Studies carried out to investigate these families led to the conclusion that most members have acquired non-redundant roles in planta. This is usually the consequence of divergent expression patterns of these isogenes and/or of different substrate specificities of the isoforms they encode. Nevertheless, much remains to be elucidated regarding the molecular bases underpinning these specificities. Protein biochemistry together with emerging quantitative proteomic technologies have then led to a better understanding of the structure of the network, which is composed of multiprotein complexes organized within the stromal compartment of plastids: whereas growing evidence suggests that the early steps of the pathway might be associated to the inner envelope membrane, several late enzymes might be localized next to the thylakoids. The question of the existence of a large integrated protein assembly channeling substrates through the whole pathway that would span the stroma remains uncertain. Finally, recent discoveries regarding the post-translational regulation of the pathway open new research horizons and may guide the development of relevant biotechnological strategies aimed at monitoring FA production in plant systems.

TOR Signaling Promotes Accumulation of BZR1 to Balance Growth with Carbon Availability in Arabidopsis.

For maintenance of cellular homeostasis, the actions of growth-promoting hormones must be attenuated when nutrient and energy become limiting. The molecular mechanisms that coordinate hormone-dependent growth responses with nutrient availability remain poorly understood in plants [1, 2]. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates nutrient and energy signaling to regulate growth and homeostasis in both animals and plants [3-7]. Here, we show that sugar signaling through TOR controls the accumulation of the brassinosteroid (BR)-signaling transcription factor BZR1, which is essential for growth promotion by multiple hormonal and environmental signals [8-11]. Starvation, caused by shifting of light-grown Arabidopsis seedlings into darkness, as well as inhibition of TOR by inducible RNAi, led to plant growth arrest and reduced expression of BR-responsive genes. The growth arrest caused by TOR inactivation was partially recovered by BR treatment and the gain-of-function mutation bzr1-1D, which causes accumulation of active forms of BZR1 [12]. Exogenous sugar promoted BZR1 accumulation and seedling growth, but such sugar effects were largely abolished by inactivation of TOR, whereas the effect of TOR inactivation on BZR1 degradation is abolished by inhibition of autophagy and by the bzr1-1D mutation. These results indicate that cellular starvation leads sequentially to TOR inactivation, autophagy, and BZR1 degradation. Such regulation of BZR1 accumulation by glucose-TOR signaling allows carbon availability to control the growth promotion hormonal programs, ensuring supply-demand balance in plant growth.

Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew.

Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway.

Sugar metabolism and the plant target of rapamycin kinase: a sweet operaTOR?

In eukaryotes, the ubiquitous TOR (target of rapamycin) kinase complexes have emerged as central regulators of cell growth and metabolism. The plant TOR complex 1 (TORC1), that contains evolutionary conserved protein partners, has been shown to be implicated in various aspects of C metabolism. Indeed Arabidopsis lines affected in the expression of TORC1 components show profound perturbations in the metabolism of several sugars, including sucrose, starch, and raffinose. Metabolite profiling experiments coupled to transcriptomic analyses of lines affected in TORC1 expression also reveal a wider deregulation of primary metabolism. Moreover recent data suggest that the kinase activity of TORC1, which controls biological outputs like mRNA translation or autophagy, is directly regulated by soluble sugars.

Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants.

Nitrate is both an important nutrient and a signalling molecule for plants. Although several components of the nitrate signalling pathway have been identified, their hierarchical organization remains unclear. Here we show that the localization of NLP7, a member of the RWP-RK transcription factor family, is regulated by nitrate via a nuclear retention mechanism. Genome-wide analyses revealed that NLP7 binds and modulates a majority of known nitrate signalling and assimilation genes. Our findings indicate that plants, like fungi and mammals, rely on similar nuclear retention mechanisms to instantaneously respond to the availability of key nutrients.

Abnormal physiological and molecular mutant phenotypes link chloroplast polynucleotide phosphorylase to the phosphorus deprivation response in Arabidopsis.

A prominent enzyme in organellar RNA metabolism is the exoribonuclease polynucleotide phosphorylase (PNPase), whose reversible activity is governed by the nucleotide diphosphate-inorganic phosphate ratio. In Chlamydomonas reinhardtii, PNPase regulates chloroplast transcript accumulation in response to phosphorus (P) starvation, and PNPase expression is repressed by the response regulator PSR1 (for PHOSPHORUS STARVATION RESPONSE1) under these conditions. Here, we investigated the role of PNPase in the Arabidopsis (Arabidopsis thaliana) P deprivation response by comparing wild-type and pnp mutant plants with respect to their morphology, metabolite profiles, and transcriptomes. We found that P-deprived pnp mutants develop aborted clusters of lateral roots, which are characterized by decreased auxin responsiveness and cell division, and exhibit cell death at the root tips. Electron microscopy revealed that the collapse of root organelles is enhanced in the pnp mutant under P deprivation and occurred with low frequency under P-replete conditions. Global analyses of metabolites and transcripts were carried out to understand the molecular bases of these altered P deprivation responses. We found that the pnp mutant expresses some elements of the deprivation response even when grown on a full nutrient medium, including altered transcript accumulation, although its total and inorganic P contents are not reduced. The pnp mutation also confers P status-independent responses, including but not limited to stress responses. Taken together, our data support the hypothesis that the activity of the chloroplast PNPase is involved in plant acclimation to P availability and that it may help maintain an appropriate balance of P metabolites even under normal growth conditions.

Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants.

Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in grape is regulated in a developmental manner in berries and leaves and by various signal molecules involved in defence such as salicylic acid, ethylene, and hydrogen peroxide. Biochemical analysis indicates that VvWRKY1 specifically interacts with the W-box in various nucleotidic contexts. Functional analysis of VvWRKY1 was performed by overexpression in tobacco, and transgenic plants exhibited reduced susceptibility to various fungi but not to viruses. These results are consistent with a possible role for VvWRKY1 in grapevine defence against fungal pathogens.

Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway.

The ripening of grape (Vitis vinifera) berry is characterized by dramatic changes in gene expression, enzymatic activities, and metabolism that lead to the production of compounds essential for berry quality. The phenylpropanoid metabolic pathway is one of the components involved in these changes. In this study, we describe the cloning and functional characterization of VvMYB5a, a cDNA isolated from a grape L. cv Cabernet Sauvignon berry library. VvMYB5a encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Expression studies in grapevine indicate that the VvMYB5a gene is mainly expressed during the early steps of berry development in skin, flesh, and seeds. Overexpression of VvMYB5a in tobacco (Nicotiana tabacum) affects the expression of structural genes controlling the synthesis of phenylpropanoid and impacts on the metabolism of anthocyanins, flavonols, tannins, and lignins. Overexpressing VvMYB5a induces a strong accumulation of several phenolic compounds, including keracyanin (cyanidin-3-rhamnoglucoside) and quercetin-3-rhamnoglucoside, which are the main anthocyanin and flavonol compounds in tobacco. In addition, VvMYB5a overexpression increases the biosynthesis of condensed tannins and alters lignin metabolism. These findings suggest that VvMYB5a may be involved in the control of different branches of the phenylpropanoid pathway in grapevine.