A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance.

Heat stress transcription factors (HSFs) and microRNAs (miRNAs) regulate different stress and developmental networks in plants. Regulatory feedback mechanisms are at the basis of these networks. Here, we report that plants improve their heat stress tolerance through HSF-mediated transcriptional regulation of MIR169 and post-transcriptional regulation of Nuclear Factor-YA (NF-YA) transcription factors. We show that HSFs recognize tomato (Solanum lycopersicum) and Arabidopsis MIR169 promoters using yeast one-hybrid/chromatin immunoprecipitation-quantitative PCR. Silencing tomato HSFs using virus-induced gene silencing (VIGS) reduced Sly-MIR169 levels and enhanced Sly-NF-YA9/A10 target expression. Further, Sly-NF-YA9/A10 VIGS knockdown tomato plants and Arabidopsis plants overexpressing At-MIR169d or At-nf-ya2 mutants showed a link with increased heat tolerance. In contrast, Arabidopsis plants overexpressing At-NF-YA2 and those expressing a non-cleavable At-NF-YA2 form (miR169d-resistant At-NF-YA2) as well as plants in which At-miR169d regulation is inhibited (miR169d mimic plants) were more sensitive to heat stress, highlighting NF-YA as a negative regulator of heat tolerance. Furthermore, post-transcriptional cleavage of NF-YA by elevated miR169 levels resulted in alleviation of the repression of the heat stress effector HSFA7 in tomato and Arabidopsis, revealing a retroactive control of HSFs by the miR169:NF-YA node. Hence, a regulatory feedback loop involving HSFs, miR169s and NF-YAs plays a critical role in the regulation of the heat stress response in tomato and Arabidopsis plants.

Landscape of the Noncoding Transcriptome Response of Two Arabidopsis Ecotypes to Phosphate Starvation.

Root architecture varies widely between species; it even varies between ecotypes of the same species, despite strong conservation of the coding portion of their genomes. By contrast, noncoding RNAs evolve rapidly between ecotypes and may control their differential responses to the environment, since several long noncoding RNAs (lncRNAs) are known to quantitatively regulate gene expression. Roots from ecotypes Columbia and Landsberg erecta of Arabidopsis (Arabidopsis thaliana) respond differently to phosphate starvation. Here, we compared transcriptomes (mRNAs, lncRNAs, and small RNAs) of root tips from these two ecotypes during early phosphate starvation. We identified thousands of lncRNAs that were largely conserved at the DNA level in these ecotypes. In contrast to coding genes, many lncRNAs were specifically transcribed in one ecotype and/or differentially expressed between ecotypes independent of phosphate availability. We further characterized these ecotype-related lncRNAs and studied their link with small interfering RNAs. Our analysis identified 675 lncRNAs differentially expressed between the two ecotypes, including antisense RNAs targeting key regulators of root-growth responses. Misregulation of several lincRNAs showed that at least two ecotype-related lncRNAs regulate primary root growth in ecotype Columbia. RNA-sequencing analysis following deregulation of lncRNA NPC48 revealed a potential link with root growth and transport functions. This exploration of the noncoding transcriptome identified ecotype-specific lncRNA-mediated regulation in root apexes. The noncoding genome may harbor further mechanisms involved in ecotype adaptation of roots to different soil environments.

Stable Inactivation of MicroRNAs in Medicago truncatula Roots.

MicroRNAs are key regulators in the development processes or stress responses in plants. In the last decade, several conserved or non-conserved microRNAs have been identified in Medicago truncatula. Different strategies leading to the inactivation of microRNAs in plants have been described. Here, we propose a protocol for an effective inactivation of microRNAs using a STTM strategy in M. truncatula transgenic roots.

A non-DNA-binding activity for the ATHB4 transcription factor in the control of vegetation proximity.

In plants, perception of vegetation proximity by phytochrome photoreceptors activates a transcriptional network that implements a set of responses to adapt to plant competition, including elongation of stems or hypocotyls. In Arabidopsis thaliana, the homeodomain-leucine zipper (HD-Zip) transcription factor ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4) regulates this and other responses, such as leaf polarity. To better understand the shade regulatory transcriptional network, we have carried out structure-function analyses of ATHB4 by overexpressing a series of truncated and mutated forms and analyzing three different responses: hypocotyl response to shade, transcriptional activity and leaf polarity. Our results indicated that ATHB4 has two physically separated molecular activities: that performed by HD-Zip, which is involved in binding to DNA-regulatory elements, and that performed by the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-associated amphiphilic repression (EAR)-containing N-terminal region, which is involved in protein-protein interaction. Whereas both activities are required to regulate leaf polarity, DNA-binding activity is not required for the regulation of the seedling responses to plant proximity, which indicates that ATHB4 works as a transcriptional cofactor in the regulation of this response. These findings suggest that transcription factors might employ alternative mechanisms of action to regulate different developmental processes.

Systemic Propagation of a Fluorescent Infectious Clone of a Polerovirus Following Inoculation by Agrobacteria and Aphids.

A fluorescent viral clone of the polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission.

Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway.

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R. solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module.

A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis.

In plants, roots are essential for water and nutrient acquisition. MicroRNAs (miRNAs) regulate their target mRNAs by transcript cleavage and/or inhibition of protein translation and are known as major post-transcriptional regulators of various developmental pathways and stress responses. In Arabidopsis thaliana, four isoforms of miR169 are encoded by 14 different genes and target diverse mRNAs, encoding subunits A of the NF-Y transcription factor complex. These miRNA isoforms and their targets have previously been linked to nutrient signalling in plants. By using mimicry constructs against different isoforms of miR169 and miR-resistant versions of NF-YA genes we analysed the role of specific miR169 isoforms in root growth and branching. We identified a regulatory node involving the particular miR169defg isoform and NF-YA2 and NF-YA10 genes that acts in the control of primary root growth. The specific expression of MIM169defg constructs altered specific cell type numbers and dimensions in the root meristem. Preventing miR169defg-regulation of NF-YA2 indirectly affected laterial root initiation. We also showed that the miR169defg isoform affects NF-YA2 transcripts both at mRNA stability and translation levels. We propose that a specific miR169 isoform and the NF-YA2 target control root architecture in Arabidopsis.

miR396 affects mycorrhization and root meristem activity in the legume Medicago truncatula.

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants.

[Non-coding RNAs involved in plant responses to environmental constraints]. / Les ARN non-codants impliqués dans la réponse des plantes aux contraintes environnementales.

In recent years, in addition to mRNAs, the non-protein-coding RNAs (or ncRNAs) have emerged as a major part of the eukaryotic transcriptome. New genomic approaches allowed the discovery of many novel long and small ncRNAs that may be linked to the generation of evolutionary complexity in multicellular organisms. Many long ncRNAs are regulated by abiotic stresses although only very few long ncRNAs have been functionally analyzed. On the other hand, small RNAs act in the regulation of gene expression at transcriptional or post-transcriptional level and several among them have been linked to abiotic stress responses. Here we describe various ncRNAs associated with environmental stress responses such as to salt, cold or nutrient deprivation. The understanding of these RNA networks may reveal novel mechanisms involved in plant adaptation to changing environmental conditions.

MicroRNAs as regulators of root development and architecture.

MicroRNAs (miRNAs) are post-transcriptional regulators of growth and development in both plants and animals. In plants, roots play essential roles in their anchorage to the soil as well as in nutrient and water uptake. In this review, we present recent advances made in the identification of miRNAs involved in embryonic root development, radial patterning, vascular tissue differentiation and formation of lateral organs (i.e., lateral and adventitious roots and symbiotic nitrogen-fixing nodules in legumes). Certain mi/siRNAs target members of the Auxin Response Factors family involved in auxin homeostasis and signalling and participate in complex regulatory loops at several crucial stages of root development. Other miRNAs target and restrict the action of various transcription factors that control root-related processes in several species. Finally, because abiotic stresses, which include nutrient or water deficiencies, generally modulate root growth and branching, we summarise the action of certain miRNAs in response to these stresses that may be involved in the adaptation of the root system architecture to the soil environment.

Small RNA diversity in plants and its impact in development.

MicroRNAs are a class of non-coding RNAs involved in post-transcriptional control of gene expression, either via degradation or translational inhibition of target mRNAs. Both experimental and computational approaches have been used to identify miRNAs and their target genes. In plants, deep sequencing methods have recently allowed the analysis of small RNA diversity in different species and/or mutants. Most sequencing efforts have been concentrated on the identification of miRNAs and their mRNA targets have been predicted based on complementarity criteria. The recent demonstration that certain plant miRNAs could act partly via inhibition of protein translation certainly opens new fields of analysis for plant miRNA function on a broader group of targets. The roles of conserved miRNAs on target mRNA stability have been analysed in different species and defined common mechanisms in development and stress responses. In contrast, much less is known about expression patterns or functions of non-conserved miRNAs. In this review, we focus on the comparative analyses of plant small RNA diversity and the action of si/miRNAs in post-transcriptional regulation of some key genes involved in root development.

Cleavage of a non-conserved target by a specific miR156 isoform in root apexes of Medicago truncatula.

Micro RNAs (miRNAs) have emerged as an important class of gene expression regulators controlling development, growth and metabolism. These short RNA molecules are 20-24 nucleotides in length and act post-transcriptionally to regulate the cleavage or translation of specific mRNA targets. In the model legume Medicago truncatula, we have recently reported identification of 100 novel and 27 conserved miRNAs in root apexes and nodules. Statistical analysis on sequencing results revealed specific miRNA isoforms for the same family (up to 3 mismatches) showing contrasting expression patterns between these tissues. Here, we report the cleavage of a non-conserved target of miR156 in root apexes complementary to a differentially expressed miR156 isoform. This suggests that changes in the abundance of miRNA isoforms may have functional consequences on the post-transcriptional regulation of new mRNA targets in different organs.

ATHB4, a regulator of shade avoidance, modulates hormone response in Arabidopsis seedlings.

Plants sense the presence of competing neighboring vegetation as a change in light quality: i.e. they sense the reduced ratio of red light to far-red light. The responses to shade are generally referred to as the shade avoidance syndrome (SAS), and involve various developmental changes intended to outgrow or outcompete the neighboring plants. Here, we analyze the function of ATHB4, a gene encoding a homeodomain-leucine zipper (HD-Zip) class-II transcription factor from Arabidopsis thaliana, the expression of which is rapidly and directly upregulated after proximity perception by the phytochrome photoreceptors. ATHB4 acts redundantly with other members of the HD-Zip class-II transcription factors. The expression of these genes is regulated by other members of the same class, forming a small transcriptional network of factors in which homeostasis is mutually controlled. Our results suggest that some members of this small gene subfamily can modulate SAS responses by controlling auxin, brassinosteroid and gibberellin molecular and/or physiological responsiveness. In particular, we propose ATHB4 as a new shade signaling component that participates in integrating shade perception and hormone-mediated growth.

Identification of primary target genes of phytochrome signaling. Early transcriptional control during shade avoidance responses in Arabidopsis.

The phytochrome (phy) photoreceptors modulate plant development after perception of light. Upon illumination of etiolated seedlings, phys initiate a transcriptional cascade by directly transducing light signals to the promoters of genes encoding regulators of morphogenesis. In light-grown plants, however, little is known about the transcriptional cascade modulated by phys in response to changes in light. The phy entry points in this cascade are completely unknown. We are particularly interested in the shade avoidance syndrome (SAS). Here we describe a subset of six genes whose expression is rapidly modulated by phys during both deetiolation and SAS in Arabidopsis (Arabidopsis thaliana). Using cycloheximide, we provide evidence that four of these phy rapidly regulated (PAR) genes are direct targets of phy signaling during SAS, revealing these genes as upstream components of the transcriptional cascade. Promoter-beta-glucuronidase fusions confirmed that PAR genes are photoregulated at the transcriptional level. Analysis of gene expression in light signal transduction mutants showed that COP1 and DET1 (but not DET2 or HY5) play a role in modulating PAR expression in response to shade in light-grown seedlings. Moreover, genetic analyses showed that one of the genes identified as a direct target of phy signaling was phy-interacting factor 3-like-1 (PIL1). PIL1 has previously been implicated in SAS in response to transient shade, but we show here that it also plays a key role in response to long-term shade. The action of PIL1 was particularly apparent in a phyB background, suggesting an important negative role for PIL1 under dense vegetation canopies.

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development.

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1.

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.

Repression of formate dehydrogenase in Solanum tuberosum increases steady-state levels of formate and accelerates the accumulation of proline in response to osmotic stress.

Formate dehydrogenase (FDH, EC 1.2.1.2.) is a soluble mitochondrial enzyme capable of oxidizing formate into CO2 in the presence of NAD+. It is abundant in non-green tissues and scarce in photosynthetic tissues. Under stress, FDH transcripts (and protein) accumulate in leaves, and leaf mitochondria acquire the ability to use formate as a respiratory substrate. In this paper, we describe the analysis of transgenic potato plants under-expressing FDH, obtained in order to understand the physiological function of FDH. Plants expressing low FDH activities were selected and the study was focused on a line (AS23) showing no detectable FDH activity. AS23 plants were morphologically indistinguishable from control plants, and grew normally under standard conditions. However, mitochondria isolated from AS23 tubers could not use formate as a respiratory substrate. Steady-state levels of formate were higher in AS23 leaves and tubers than in control plants. Tubers of untransformed plants oxidized 14C formate into 14CO2 but AS23 tubers accumulated it. In order to reveal a possible phenotype under stress conditions, control and AS23 plants were submitted to drought and cold. These treatments dramatically induced FDH transcripts in control plants but, whatever the growth conditions, no 1.4 kb FDH transcripts were detected in leaves of AS23 plants. Amongst various biochemical and molecular differences between stressed AS23 and control plants, the most striking was a dramatically faster accumulation of proline in the leaves of drought-stressed plants under-expressing FDH.