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International audience; When a plant germinates in the soil, elongation of stem-like organs is enhanced whereas leaf and root growth is inhibited. How these differential growth responses are orchestrated by light and integrated at the organismal level to shape the plant remains to be elucidated. Here, we show that light signals through the master photomorphogenesis repressor COP1 to coordinate root and shoot growth in Arabidopsis. In the shoot, COP1 regulates shoot-to-root auxin transport by controlling the transcription of the auxin efflux carrier gene PIN-FORMED1 (PIN1), thus appropriately tuning shoot-derived auxin levels in the root. This in turn directly influences root elongation and adapts auxin transport and cell proliferation in the root apical meristem by modulating PIN1 and PIN2 intracellular distribution in the root in a COP1-dependent fashion, thus permitting a rapid and precise tuning of root growth to the light environment. Our data identify auxin as a long-distance signal in developmental adaptation to light and illustrate how spatially separated control mechanisms can converge on the same signaling system to coordinate development at the whole plant level.

Osmotic stress critically limits plant growth and crop productivity. The identification of genes underlying the mechanisms of stress response is the subject of intense research in plant biology. Through microarray analyses we previously identified and isolated RGGA gene, coding for an RNA binding protein, whose expression was specifically induced in Solanumtuberosum cell cultures gradually exposed to osmotic stress. The aim of this study was to confirm the role of RGGA as a functional RNA binding protein required for a proper response to stress condition. We identified the RGGA orthologue in Arabidopsis thaliana (AtRGGA, At4g16830) and evaluated the influence of drought and salt stress on AtRGGA gene expression in cells and seedlings exposed to high concentrations of NaCl, PolyEthyleneGlycole (PEG) and abscisic acid (ABA).Interestingly, AtRGGA gene expression was up-regulated in seedlings after long-term exposure to NaCl and PEG, while short-term treatments with NaCl resulted in AtRGGA down- regulation. To investigate the protein sub-cellular localization, a YFP-RGGA fusion protein was used. Fluorescence signal indicated that RGGA is localized in the cytoplasm and the peri- nuclear region. In addition, a ?-glucuronidase (GUS) assay showed promoter activity in several tissues, including guard cells of stomata, the organs controlling transpiration. Electrophoresis Mobility Shift Assays with total RNA using recombinant His-RGGA clearly showed that RGGA is capable of binding RNA in vitro. To carry out a functional analysis, a gain- and loss-of-function approach was performed using rggaknock-out mutants and AtRGGA over-expressing plants. In addition, rggaknock-out mutant was hyper-sensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. Accordingly, the over- expressing plants showed a higher tolerance both in vitro and in soil and accumulated lower levels of proline when exposed to salt and drought stress conditions. Finally, a global analysis of gene expression using microarrays, revealed extensive alterations in the transcriptome of AtRGGA over-expressing plants and rgga mutants under osmotic stress, indicating that RGGA participates in the modulation of transcript abundance of several key genes involved in abiotic stress response. The data taken together provide compelling evidence that the RGGA gene is involved in important mechanisms of plant response to osmotic stress.

Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant,Arabidopsis thaliana. REGIA involves: 1. the definition ofTFgene expression patterns inArabidopsis; 2. the identification of mutations atTFloci; 3. the ectopic expression of TFs (or derivatives) inArabidopsisand in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both aTFgene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme.

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and BUSHY AND DWARF2 (BUD2), is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper (HD-ZIP) III and key auxin signalling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein ARABIDOPSIS THALIANA HOMEOBOX8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signalling genes such as LIKE AUXIN RESISTANT2 (LAX2) and LAX3. We propose that proper formation and differentiation of xylem depends on the balance between positive and negative feedback loops operating through HD-ZIP III genes.

Spinal muscular atrophy (SMA) is a neuromuscular disorder and one of the most common genetic causes of infant mortality. The disease is characterized by a selective degeneration of lower spinal cord motor neurons, which leads to progressive muscle atrophy and death. SMA is caused by mutations of the survival of motor neuron gene, Smn1, which is ubiquitously expressed. Although the genetic bases of SMA have been extensively studied, it is still unknown how the absence of Smn1 induces the selective degeneration of motor neurons, and which are the molecular mechanisms that underlie the disease. This contributes to the lack of an effective treatment being developed. Unbiased chemical screens can be performed in vivo using small animal model organisms, and contribute to identify potential therapeutic compounds as well as elucidate the molecular basis of the disease. In C.elegans two SMA models have been developed by classical genetic approaches: the smn-1(ok355) null mutant (Briese et al., Hum. Mol. Gen. 2009), in which the loss of smn-1 produces pleiotropic phenotypes and lethality, and the smn-1(cb131) hypomorphic mutant (Sleigh et al., Hum. Mol. Gen. 2010), which displays similar but milder defects. However no morphological alteration in the nervous system and no variations in the number of motor neurons have been detected in these genetic mutants. The hypomorphic mutant has been used to screen a chemical library and has led to the identification of some compounds capable of improving at least one of the disease phenotypes of the model. In order to find molecules with a protective role against the neurodegeneration caused by smn-1 loss, we took advantage of a genetic model developed in our laboratory, which is based on neuron-specific RNAi of smn-1 (Esposito et al., Gene 2007). Transgenic strains in which smn-1 is knocked down specifically in the GABAergic motor neurons present an age-dependent neurodegeneration, which results in altered backward movement and in neuronal cell death. Importantly, these animals are viable and fertile allowing us to overcome the lethality problem related to the other C.elegans SMA model carrying smn-1 loss of function. We initially tested on this model a panel of chemical compounds that were selected based on their known function. We have confirmed that valproic acid, a compound successfully used also in mice SMA models (Seo et al., Biochim. Biophys. Acta, 2013), can partially prevent neuronal death in C. elegans. Then, we found that the Ca2+-chelating agent EGTA has a protective function on neurodegeneration, suggesting that Ca2+ plays a role in smn-1-induced GABAergic cell death. Moreover, we have tested the effects of natural compounds and have obtained interesting results with juices of broccoli sprouts, which are currently under characterization. Finally, we are setting up the conditions to carry out an automated screen of a chemical library, consisting of 1280 FDA-approved compounds.

Plants have evolved sophisticated mechanisms to sense the presence of other plants growing nearby and adjust their growth rate accordingly. The early perception of neighbor proximity depends on the detection of light quality changes. Within a vegetation community, the ratio of red (R) to far-red (FR) light is lowered by the absorption of R light by photosynthetic pigments. This light quality change is perceived through phytochrome (phyB, phyD and phyE in Arabidopsis) as a signal of the proximity of neighbors, and induces a suite of developmental responses (termed the shade avoidance response). In Arabidopsis shade avoidance is regulated by a balance of positive (PIF) and negative (HFR1/SICS1) regulators of gene expression which ensures a fast reshaping of the plant body towards an environment optimal for growth while at the same time avoiding an exaggerated reaction to low R/FR. Persistency of a low R/FR signal enhances the activity of phyA and, in turn, of HY5, a master regulator of seedling de-etiolation. Several hormones, such as gibberellins and brassinosteroids have been implicated in shade-induced elongation. However, a compelling amount of evidence indicates that low R/FR-induced changes in auxin homeostasis and auxin transport are central in the shade avoidance response. This chapter describes the recent advances in understanding how auxin coordinates plant growth in a low R/FR light environment.

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS 5 (ACL5) and BUSHY AND DWARF 2 (BUD2)is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of the homeodomain leucine zipper (HD-Zip) III and key auxin signalling geens. This mechanism involves the posititve regulation of thermospermine biosynthesis by the HD-Zip III protein ARABIDOPSIS THALIANA HOMEOBOX tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-Zip III protein REVOLUTA contributes to the increased leaf vascularizaton and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signalling genes such as LIKE AUXIN RESISTANT 2 (LAX2) and (LAX3). Through this work we present a model of positive and negative feedback between auxin and HD-Zip gene expressions that tunes the rate of xylem differentiation.

Development of the vascular system in higher plants is a fundamental process that affects plant growth and yield. In addition, xylem production per se has an enormous economic impact. In fact, efficient water utilization, wood and fiber industry, biofuel production, food and feed digestibility largely depend on differentiation of lignified elements in the vascular apparatus specialized for water transport and mechanical support. The HD-ZIP III family of transcription factors is one of the most important and well characterized player that control vascular development (Myashima et al., 2012). ATHB8, in particular, is a marker of preprocambial cell state that accurately predicts sites of leaf vein formation and promotes proliferation and differentiation of xylem cells (Baima et al., 2001; Donner et al., 2009). Recently, the ACAULIS5 (ACL5) gene, encoding a polyamine synthase, has been involved in some aspects of xylem differentiation; it has been proposed that its product thermospermine, an isomer of spermine, is important to prevent premature cell death of tracheary elements (Knott et al., 2007; Muniz et al., 2008). In addition, it has been previously demonstrated that the acl5 mutant, also known as thickvein, causes the formation of an increased number of veins and vascular elements in leaves and stems (Hanzawa et al., 1997, 2000; Clay et al., 2005), likely by altering auxin signaling (Yoshimoto et al., 2012). More recently, taking advantage of several complementary experimental approaches, including ChIP, we demonstrated that ACL5 is directly regulated in a positive manner by ATHB8. We also proposed a model in which ATHB8 and other HD-ZIPIII transcription factors and ACL5 establish a negative feedback loop, likely connected to auxin, necessary for proper regulation of vascular cell differentiation (Baima et al., submitted). In the course of these studies, we found that transgenic plants expressing high levels of ACL5 in xylem precursor cells significantly delay or completely inhibit the differentiation of these cells into tracheary elements in leaf as well as in primary and secondary vasculature of stem and hypocotyl. Consistent with the proposed regulatory loop, gene expression analysis revealed that ATHB8 expression is down-regulated in these transgenic plants. To determine which genes involved in xylem cell differentiation are regulated by the ATHB8/ACL5 module, the transcriptome of transgenic lines enriched in undifferentiated xylem cells was compared to that of plants displaying a wild-type vasculature. The results of this analysis will be presented.

The shade avoidance syndrome (SAS) refers to a set of plant responses initiated after perception by the phytochromes of light with a reduced red to far-red ratio, indicative of vegetation proximity or shade. These responses, including elongation growth, anticipate eventual shading from potential competitor vegetation by overgrowing neighboring plants or flowering to ensure production of viable seeds for the next generation. In Arabidopsis thaliana seedlings, the SAS includes dramatic changes in gene expression, such as induction of PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), encoding an atypical basic helix-loop-helix (bHLH) protein that acts as a transcriptional co-factor to repress hypocotyl elongation. Indeed, PAR1 has been proposed to act fundamentally as a dominant negative antagonist of conventional bHLH transcription factors by forming heterodimers with them to prevent their binding to DNA or other transcription factors. Here we report the identification of PAR1-interacting factors, including the brassinosteroid signaling components BR-ENHANCED EXPRESSION (BEE) and BES1-INTERACTING MYC-LIKE (BIM), and characterize their role as networked positive regulators of SAS hypocotyl responses. We provide genetic evidence that these bHLH transcriptional regulators not only control plant growth and development under shade and non-shade conditions, but are also redundant in the control of plant viability. Our results suggest that SAS responses are initiated as a consequence of a new balance of transcriptional regulators within the pre-existing bHLH network triggered by plant proximity, eventually causing hypocotyls to elongate., Fellowships or contracts were provided by CSIC (J.B.–T. and M.S.–M.), the Ministerio de Educación (A.G.), the Ministerio de Economía y Competitividad (I.R.–V, and M.G.) and the Gobierno de Chile (N.C.–E.). Research in our laboratories is supported by grants to I.R. from the Italian Ministry of Economy and Finance (Project FaReBio di Qualità) and to J.F.M.–G.'s laboratory (XRB, 2009-SGR697 from the Generalitat de Catalunya and CSD2007-00036, BIO2005-00154, BIO2008-00169 and BIO2011-23489 from Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional funds).

CCCH zinc finger domain consists of a sequence with three cysteines and one histidine residues with strictly defined spacing: C-X4-15-C-X4-6-C-X3-H. As in animals, even in plants this domain has been found in some RNA-binding proteins involved in several important biological processes. A genome-wide analysis has identified 68 and 67 CCCH family genes in Arabidopsis and rice, respectively, these genes are divided in several sub-family, among them the Tandem Zinc Finger (TZF) gene subfamily contains a unique TZF motif that can only be found in plants, and its members are involved in stress response. TZF genes are represented throughout the evolutionary scale, from algae to higher plants. The phylogenetic analysis revealed that zinc-finger domain is strongly conserved from algae to higher plants, suggesting that these domains have a primary importance for the molecular function of these proteins. Our attention was focused on a Triticum durum TZF protein, TdTZF1-A, and its homeologous gene, TdTZF1-B. Full length sequences of TdTZF1-A (characterized as responsive to cold and dehydration stresses) and TdTZF1-B genes were obtained from the Creso variety and they were found to correspond to homoelogous genes positioned on chromosomes 3A and 3B respectively. Among Arabidopsis TZF genes, AtTZF3 is the putative ortholog of TdTZF1-A, and to carryout a functional study we created under- and over-expression mutants for this gene. A deep phenotipic evaluation of the germination process in presence of ABA was performed. AtTZF3 silenced lines showed a better germination capacity in ABA treatments than wilde-type. Conversely, overexpressing lines have a greater difficulty to germinate in the same conditions. Similar results are obtained under salt stress. These data suggest that AtTZF3 is a negative regulator of seed germination. Expression analysis under stress conditions and germination suggests the involvement of TdTZF1-A and TdTZF1-B in the regulation of seed germination in durum wheat.

The success of competitive interactions between plants determines the chance of survival of individuals and eventually of whole plant species. Shade-tolerant plants have adapted their photosynthesis to function optimally under low light conditions. These plants are therefore capable of long-term survival under a canopy shade. In contrast, shade-avoiding plants adapt their growth to perceive maximum sunlight and therefore rapidly dominate gaps in a canopy. Daylight contains roughly equal proportions of red (R) and far-red (FR) light, but within vegetation that ratio is lowered as a result of R absorption by photosynthetic pigments. This light quality change is perceived through the phytochrome system as an unambiguous signal of the proximity of neighbours resulting in the shade avoidance response. This adaptive reaction is achieved by a set of responses including enhanced internode and petiole extension growth, increased apical dominance, retarded leaf development, and an acceleration of flowering. Genomic and genetic analyses by a number of laboratories including ours have identified several low R/FR regulated genes and key regulators involved in the shade avoidance response. However, very little is known about the cascade of events triggered by low R/FR that give rise to full activation of the response and eventually lead to adaptation to an unfavourable light environment. By combining genome-wide experimental and computational analyses in wild type and genetically altered plants exposed to low R/FR light for different times, we identified novel regulatory circuits controlling plant adaptation to canopy shade.

In the last decades several genes that regulate abiotic stress response were identified in the model plant Arabidopsis. The completion of the Arabidopsis genome sequence in 2000, and the development of molecular high throughput 'omics' platforms that allow the processing of several samples has speed up the simultaneous functional characterization of genes that regulate the abiotic stress tolerance in plants. The fundamental molecular mechanisms that underlie the plant abiotic responses are quite conserved among plant species therefore the knowledge gained in the model plant Arabidopsis can be exploited to improve stress tolerance in crop plants. Many examples demonstrate that ectopic expression of key genes involved in the stress response can induce stress tolerance in different crops demonstrating the potential of the translational approaches. Nevertheless the generation of crops that have improved tolerance in field conditions is still a challenge. However the omics approach in crop species can certainly contribute in the understanding of the interplay between response pathways to different abiotic stresses, condition that is found in typical open fields.

When a plant germinates in the soil, elongation of stem-like organs is enhanced whereas leaf and root growth is inhibited. How these differential growth responses are orchestrated by light and integrated at the organismal level to shape the plant remains to be elucidated. Here, we show that light signals through the master photomorphogenesis repressor COP1 to coordinate root and shoot growth in Arabidopsis. In the shoot, COP1 regulates shoot-to-root auxin transport by controlling the transcription of the auxin efflux carrier gene PIN-FORMED1 (PIN1), thus appropriately tuning shoot-derived auxin levels in the root. This in turn directly influences root elongation and adapts auxin transport and cell proliferation in the root apical meristem by modulating PIN1 and PIN2 intracellular distribution in the root in a COP1-dependent fashion, thus permitting a rapid and precise tuning of root growth to the light environment. Our data identify auxin as a long-distance signal in developmental adaptation to light and illustrate how spatially separated control mechanisms can converge on the same signaling system to coordinate development at the whole plant level.

The acaulis5 (acl5) loss-of-function mutant, also known as thickvein, is dwarf and characterized by the formation of an increased number of veins and vascular elements in leaves and stems (Hanzawa et al., 1997, 2000; Clay et al., 2005). The ACL5 gene, encoding a polyamine synthase producing thermospermine, an isomer of spermine, has been proposed to control xylem specification by preventing premature cell death of tracheary elements (Knott et al., 2007; Muniz et al., 2008). Interestingly, we found that higher levels of ACL5, likely resulting in increased thermospermine concentration, significantly delay or completely inhibit the differentiation of procambial cells into tracheary elements in leaf as well as in primary and secondary vasculature of stem and hypocotyl. Consistently with the up-regulation shown in acl5 (Imai et al. 2006; Kakhei et al., 2008), we found a down-regulation of the expression of the HD-ZIPIII transcription factor ATHB8, a marker of preprocambial cell state that accurately predicts sites of leaf vein formation and promotes proliferation and differentiation of vascular precursor cells (Baima et al., 2001; Donner et al., 2009), in plants with increased ACL5 expression. These observations suggest that the vascular defects observed in both mutant and over-expressing plants may, at least in part, depend on the level of HD-ZIPIII activity. Indeed, a careful genetic analysis of acl5 in combination with athb8 and other hd-zipiii mutants indicated that some of the developmental defects caused by the lack of ACL5 activity are abolished by the loss of HD-ZIPIII transcription factors. On the other hand, we could demonstrate by different approaches, including ChIP, that ACL5 is directly regulated in a positive manner by ATHB8. These findings have been integrated in a model in which HD-ZIPIII and ACL5 genes establish a negative feedback loop necessary for proper regulation of vascular development.

The success of competitive interactions between plants determines the chance of survival of individuals and eventually of whole plant species. Shade-tolerant plants have adapted their photosynthesis to function optimally under low-light conditions. These plants are therefore capable of long-term survival under a canopy shade. In contrast, shade-avoiding plants adapt their growth to perceive maximum sunlight and therefore rapidly dominate gaps in a canopy. Daylight contains roughly equal proportions of red (R) and far-red (FR) light, but within vegetation that ratio is lowered as a result of the R absorption by photosynthetic pigments. This light quality change is perceived through the phytochrome system as an unambiguous signal of the proximity of neighbours resulting in the shade avoidance response. This adaptive reaction is achieved by a set of responses including enhanced internode and petiole extension growth, increased apical dominance, retarded leaf development, and an acceleration of flowering. However, if a plant succeeds in the attempt to overgrow its neighbours and the photosynthetic organs perceive daylight again, the shade avoidance response is rapidly switched off through phytochrome photoconversion. The adaptive responses result in changes in the distribution of assimilates between leaves, stems, and roots. Genomic and genetic analyses by our and other laboratories have identified several low R/FR-regulated genes and key regulators involved in the shade avoidance response. However, very little is known about the cascade of events triggered by low R/FR that give rise to the full activation of the response and, later on, to the adaptation process when a plant does not succeed to overgrow its neighbours. Therefore, shade avoidance response was examined by genome wide expression profiling in wild type and genetically altered plants exposed to low R/FR light for different times. To identify gene networks, both computational and experimental approaches are being pursued. Informatic analyses provided insights into functional clusters and their dynamics, predictions of cis-regulatory elements for genes temporally regulated during shade avoidance response, inference of gene regulatory interactions. Together, these analyses uncovered novel aspects of shade avoidance, and generated testable hypotheses on gene regulatory circuitry underlying plant responses to light quality changes.

CCCH zinc finger domain consists of a sequence with three cysteines and one histidine residues with strictly defined spacing: C-X4-15-C-X4-6-C-X3-H. First identified in proteins of Tristetraprolin family in mammals, involved in regulation of stability of cytokine mRNAs, this domain has been found in plant RNA-binding proteins involved in the control of important biological processes such as floral reproductive organ identity determination and calmodulinmediated RNA processing. A gene coding for a CCCH zinc finger protein, named 2H8, was isolated in durum wheat and characterised as responsive to cold and dehydration stresses (De Leonardis et al., 2007). Six additional cDNA sequences were identified in the wheat EST database following a similarity search. These genes were characterised by expression studies under cold and water stress conditions. Overall, a family of more than sixty genes coding for CCCH proteins was recently described in Arabidopsis. A functional conservation between a sub-group of stress-related Arabidopsis CCCH genes and the seven highly homologous genes, identified in durum wheat as responsive to cold and drought stresses has been suggested. To gain information of the role of this gene family in stress response, a functional analysis on the Arabidopsis genes via mutant analysis is underway. In particular, this study is focused on the characterization of a set of Arabidopsis lines generated by ihpRNA interference and amiRNA technologies resulting in the down-regulation of the CCCH zinc finger gene At4g29190 (the most homologous to the 2H8 gene of wheat). A deep phenotypic evaluation of the germination process under abiotic stress conditions revealed that the mutant lines grow better than the wild type. These results suggest that the product of the At4g29190 gene operates as a negative regulator of germination in particular in presence of salt stress and low temperature. Experiments are in progress to test whether At4g29190 regulates the expression of the genes controlling the accumulation of GA and ABA genes whose activity is important in the germination process. A detailed expression analysis of the homologous wheat gene is also in progress to verify a possible involvement of this gene product in the germination of wheat seeds under stress conditions.

A plant growing in the field has the unique ability to sense the presence of other plants growing nearby and adjust its growth rate accordingly. The early perception of neighbour proximity depends on the detection of light quality changes. Within a vegetation community, the ratio of red (R) to far-red (FR) light is lowered by the absorption of R light by photosynthetic pigments. This light quality change is perceived through the phytochrome system as an unambiguous signal of the proximity of neighbours, and induces a suite of developmental responses (termed the shade avoidance response) that, when successful, result in the overgrowth of those neighbours. The shade avoidance response is a strategy of major adaptive significance to plants in natural communities. However, since elongation is often achieved at the expense of leaf and root growth, and, in the long term, low R/FR exposure leads to early flowering with a reduced seed set, shade avoidance can significantly impact yield in high-density crop plantings. Work performed by several laboratories, including ours, identified several key transcription factor genes that underpin shade avoidance and uncovered complex molecular interactions between low R/FR light and hormone signalling pathways. Among hormones, auxin seems to have a central role in several aspects of shade avoidance response. Many lines of evidence connect this hormone to the rapid elongation response provoked by light quality changes. Furthermore, our recent work establishes that auxin plays a pivotal role in leaf and root responses to low R/FR as well, suggesting that this hormone may act as a coordinator of plant growth responses to environmental light quality changes. Shade avoidance is a highly dynamic response. If the plant succeeds in the attempt to overgrow its neighbours and the photosynthetic organs perceive daylight again, the shade avoidance response is rapidly reverted through phytochrome photoconversion. On the other hand, persistent low R/FR signalling triggers mechanisms that prevent an exaggerated reaction when the plant is unsuccessful in escaping canopy shade. HFR1/SICS1, a bHLH transcription factor, plays a central role in the attenuation of virtually all plant responses to canopy shade. Our recent data demonstrate that HFR1/SICS1 functions in the phyB signal transduction pathway, and acts in concert with other transcription factors modulated through phyA in the adaptation of the plant to low R/FR environments.

The success of competitive interactions between plants determines the chance of survival of individuals and eventually of whole plant species. Shade-tolerant plants have adapted their photosynthesis to function optimally under low-light conditions. These plants are therefore capable of long-term survival under a canopy shade. In contrast, shade-avoiding plants adapt their growth to perceive maximum sunlight and therefore rapidly dominate gaps in a canopy. Daylight contains roughly equal proportions of red (R) and far-red (FR) light, but within vegetation that ratio is lowered as a result of the R absorption by photosynthetic pigments. This light quality change is perceived through the phytochrome system as an unambiguous signal of the proximity of neighbours resulting in the shade avoidance response. This adaptive reaction is achieved by a set of responses including enhanced internode and petiole extension growth, increased apical dominance, retarded leaf development, and an acceleration of flowering. However, if a plant succeeds in the attempt to overgrow its neighbours and the photosynthetic organs perceive daylight again, the shade avoidance response is rapidly switched off through phytochrome photoconversion. The adaptive responses result in changes in the distribution of assimilates between leaves, stems, and roots. Genomic and genetic analyses by our and other laboratories have identified several low R/FRregulated genes and key regulators involved in the shade avoidance response. However, very little is known about the cascade of events triggered by low R/FR that give rise to the full activation of the response and, later on, to the adaptation process when a plant does not succeed to overgrow its neighbours. Therefore, shade avoidance response was examined by genome wide expression profiling in wild type and genetically altered plants exposed to low R/FR light for different times. To identify gene networks, both computational and experimental approaches are being pursued. Informatic analyses provided insights into functional clusters and their dynamics, predictions of cisregulatory elements for genes temporally regulated during shade avoidance response, inference of gene regulatory interactions. Together, these analyses uncovered novel aspects of shade avoidance, and generated testable hypotheses on gene regulatory circuitry underlying plant responses to light quality changes. This work was supported by a grant from MiPAAF - AGRONANOTECH Program.

The shade avoidance response is a strategy of major adaptive significance to plants in natural communities. It is highly widespread in the angiosperms, and depends on the ability of the plant to perceive the presence of neighbors. Within vegetation, the ratio of red to far-red (R/FR) is lowered by the absorption of R light by photosynthetic pigments. This light quality change is perceived through the phytochrome system as an unambiguous signal of the proximity of neighbors. Upon sensing a low R/FR ratio, a shade-avoiding plant reacts very rapidly and enhances elongation growth even before it is directly shaded. If the plant succeeds in the attempt to overgrow its neighbors and the photosynthetic organs perceive daylight again, the shade avoidance response is rapidly reverted through phytochrome photoconversion. Consistent with the rapidity of plant response to low R/FR and its reversibility upon perception of high R/FR, changes in gene expression are rapid and reversible. The transcript level of the Arabidopsis HD-Zip ATHB2 and bHLH PIL1 transcription factor genes, functionally implicated in shade avoidance response, increases within a few minutes of low R/FR exposure. Significantly, ATHB2 and PIL1 transcript levels fall very rapidly after transfer from low to high R/FR. Low R/FR also provokes a rapid induction of the Arabidopsis HFR1/SICS1 gene, a negative controller of the shade avoidance response, ensuring that an exaggerated reaction does not occur when the plant is unsuccessful in escaping canopy shade. In this unfavorable environmental condition, HFR1/SICS1 is likely to play a fundamental role in the acclimation of the plant, and by delaying flowering, to ensure a better seed production needed for long term survival. Recent work revealed that the same low R/FR signal that induces hypocotyl elongation also triggers a rapid arrest of leaf primordium growth ensuring that plant resources are redirected into extension growth. The growth arrest induced by low R/FR depends on auxin-induced cytokinin breakdown in incipient vein cells of developing primordia, thus demonstrating the existence of a previously unrecognized regulatory circuit underlying plant response to canopy shade.

Plants have evolved sophisticated sensing mechanisms that operate through phytochromes, perceiving changes in the red:far-red ratio, which trigger morphological changes to avoid shade. The shade-avoidance response essentially redirects resources and growth potential from the leaf and storage organs into increased extension growth to optimize light capture by plants. Recent studies implicate ATHB-2, a homeodomain-leucine zipper transcription factor, as a regulator of shade-avoidance responses and establish a strong link between this factor and auxin signaling. The action of ATHB-2 is likely to generate changes in auxin distribution that produce distinct but coordinated effects on different cell types across the plant. Future studies should highlight how polarity of auxin transport is altered in response to light-quality changes.

GLABRA2 (GL2)/ATHB-10 encodes a homeodomain protein that belongs to the homeodomain-leucine zipper family. Mutant studies have revealed that this gene is involved in trichome, root-hair and seed-coat development. We used reverse genetics to investigate the role of GL2 in trichome development. A transgene consisting of a GL2-coding fragment preceded by the cauliflower mosaic virus 35S promoter (35S::GL2) did not complement defects in the gl2-1 mutant. In the wild-type genetic background, 35S::GL2 caused gl2-mutant-like and scarcely viable phenotypes, suggesting that ectopic overexpression of GL2 interrupts endogenous GL2 function in trichome development and is toxic to plants. On the other hand, another GL2 transgene containing the GL2 promoter (pGL2::GL2) complemented the gl2-1 mutation. Entopically additive expression of GL2 by introduction of pGL2::GL2 in the wild-type genetic background noticably increased the number of trichomes and induced production of adjacent trichomes. Consistent with this result, gl2-1/+ heterozygous leaves, whose GL2 expression was expected to decrease, had fewer trichomes than +/+ leaves. These results indicate that GL2 quantitatively regulates the frequency of trichome initiation and is involved in determining trichome spacing.

The Arabidopsis homeobox gene ATHB-2 is tightly regulated by light signals, and is thought to direct morphological changes during shade avoidance responses. To understand how ATHB-2 mediates light signals in plant morphogenesis, we investigated its transcriptional network. We constructed a gene encoding a chimeric transcription factor (HD-Zip-2-V-G) that is expected to activate target genes of ATHB-2 in a glucocorticoid-dependent manner. In transgenic Arabidopsis plants expressing HD-Zip-2-V-G, glucocorticoid treatment activates the ATHB-2 gene itself, independent of de novo protein synthesis. An in vitro DNase I-footprinting experiment showed that recombinant ATHB-2 protein specifically bound to an ATHB-2 promoter region. These complementary results indicate that ATHB-2 recognizes its own promoter. Consistent with the fact that ATHB-2 itself has been shown to act as a repressor, expression of the endogenous ATHB-2 gene was repressed in transgenic plants overexpressing an ATHB-2 transgene. Moreover, target-gene analysis using the HD-Zip-2-V-G suggested that ATHB-2 recognizes other HD-Zip II subfamily genes. We conclude that ATHB-2 has a negative autoregulatory loop and may be involved in a complicated transcriptional network involving paralogous genes, as in the case with animal homeobox genes.

We have modified the biolistic process to establish a transient assay for rapid analysis of transcription factors in arabidopsis. ATHB-1, an arabidopsis HD-Zip protein, was used as a transacting factor in particle bombardment experiments with arabidopsis leaves, and was found to transactivate a reporter gene in a binding site-specific manner. The experiments show the feasibility of using microprojectile-mediated gene transfer for rapid functional analysis of transcription factors in arabidopsis

Homeodomain-leucine zipper (HD-Zip) proteins are putative transcription factors identified only in plants. The study of the DNA-binding properties of the ATHB-1 and -2 HD-Zip (HD-Zip-l and -2) domains showed that they interact with DNA as homodimers and recognize two distinct 9 bp pseudopalindromic sequences, CAAT(A/T)ATTG (BS-1) and CAAT(G/C)ATTG (BS-2), respectively, as determined by selecting high-affinity binding sites from random-sequence DNA. Here, we report a mutational analysis of the HD-Zip-2 domain. We determined that conserved amino acid residues of helix 3, Val47 and Asn51, and Arg55 are essential for the DNA-binding activity of the HD-Zip-2 domain. We demonstrated that the preferential recognition of a G/C base-pair at the central position by the HD-Zip-2 domain is abolished either by the replacement of Arg55 with lysine or by the substitution of Glu46 and Thr56 with the corresponding residues of the HD-Zip-l domain (alanine and tryptophan, respectively). In contrast, substitution of Arg55 with lysine in the HD-Zip-3 domain significantly reduced DNA-binding activity without changing the specificity of recognition. Finally, we determined that differences in residues outside helix 3 further contribute to the DNA-binding specificity of the HD-Zip domain. Taken together, the data strongly suggest that the preferential recognition of BS-2 and -1 by the HD-Zip-2 and -1, domains, respectively, may be attributable to a distinct orientation of the side-chain of Arg55 in these two domains.