Javier Paz-Ares

The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses

This work identifies two transcription factors, MYC3 and MYC4, as targets of JAZ repressors and regulators of responses to jasmonate. It finds a specificity of transcription factor activity that could be a clue to understanding the diversity of JA-regulated responses. Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

MYB transcription factors in plants

The cloning of the first transcription factor from plants, the C1 gene of maize, indicated that plants use transcription factors that are structurally related to those of animals in their control of gene expression, because C1 showed significant structural homology to the vertebrate cellular proto-oncogene c-MYB. Since 1987, the catalogue of MYB-related transcription factors has increased considerably in size due, primarily, to the ever-expanding number of MYB genes identified in higher plants (Arabidopsis thaliana is estimated to contain more than a hundred MYB genes). In vertebrates, the MYB-related proto-oncogenes comprise a small family with a central role in controlling cellular proliferation and commitment to development. However, while the functions of some plant MYB genes are relatively well understood they are, at present, quite distinct from their animal counterparts.

A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis.

Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress.

A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ∼20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

Interaction between Phosphate-Starvation, Sugar, and Cytokinin Signaling in Arabidopsis and the Roles of Cytokinin Receptors CRE1/AHK4 and AHK3

Cytokinins control key processes during plant growth and development, and cytokinin receptors CYTOKININ RESPONSE 1/WOODEN LEG/ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/WOL/AHK4), AHK2, and AHK3 have been shown to play a crucial role in this control. The involvement of cytokinins in signaling the status of several nutrients, such as sugar, nitrogen, sulfur, and phosphate (Pi), has also been highlighted, although the full physiological relevance of this role remains unclear. To gain further insights into this aspect of cytokinin action, we characterized a mutant with reduced sensitivity to cytokinin repression of a Pi starvation-responsive reporter gene and show it corresponds to AHK3. As expected, ahk3 displayed reduced responsiveness to cytokinin in callus proliferation and plant growth assays. In addition, ahk3 showed reduced cytokinin repression of several Pi starvation-responsive genes and increased sucrose sensitivity. These effects of the ahk3 mutation were especially evident in combination with the cre1 mutation, indicating partial functional redundancy between these receptors. We examined the effect of these mutations on Pi-starvation responses and found that the double mutant is not significantly affected in long-distance systemic repression of these responses. Remarkably, we found that expression of many Pi-responsive genes is stimulated by sucrose in shoots and to a lesser extent in roots, and the sugar effect in shoots of Pi-starved plants was particularly enhanced in the cre1 ahk3 double mutant. Altogether, these results indicate the existence of multidirectional cross regulation between cytokinin, sugar, and Pi-starvation signaling, thus underlining the role of cytokinin signaling in nutrient sensing and the relative importance of Pi-starvation signaling in the control of plant metabolism and development.

Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida.

The MYB.Ph3 protein recognized two DNA sequences that resemble the two known types of MYB DNA binding site: consensus I (MBSI), aaaAaaC(G/C)‐GTTA, and consensus II (MBSII), aaaAGTTAGTTA. Optimal MBSI was recognized by animal c‐MYB and not by Am305 from Antirrhinum, whereas MBSII showed the reverse behaviour. Different constraints on MYB.Ph3 binding to the two classes of sequences were demonstrated. DNA binding studies with mutated MBSI and MBSII and hydroxyl radical footprinting analysis, pointed to the N‐terminal MYB repeat (R2) as the most involved in determining the dual DNA binding specificity of MYB.Ph3 and supported the idea that binding to MBSI and MBSII does not involve alternative orientations of the two repeats of MYB.Ph3. Minimal promoters containing either MBSI and MBSII were activated to the same extent by MYB.Ph3 in yeast, indicating that both types of binding site can be functionally equivalent. MYB.Ph3 binding sites are present in the promoter of flavonoid biosynthetic genes, such as the Petunia chsJ gene, which was transcriptionally activated by MYB.Ph3 in tobacco protoplasts. MYB.Ph3 was immunolocalized in the epidermal cell layer of petals, where flavonoid biosynthetic genes are actively expressed. This strongly suggests a role for MYB.Ph3 in the regulation of flavonoid biosynthesis.

Plant hormones and nutrient signaling

Plants count on a wide variety of metabolic, physiological, and developmental responses to adapt their growth to variations in mineral nutrient availability. To react to such variations plants have evolved complex sensing and signaling mechanisms that allow them to monitor the external and internal concentration of each of these nutrients, both in absolute terms and also relatively to the status of other nutrients. Recent evidence has shown that hormones participate in the control of these regulatory networks. Conversely, mineral nutrient conditions influence hormone biosynthesis, further supporting close interrelation between hormonal stimuli and nutritional homeostasis. In this review, we summarize these evidences and analyze possible transcriptional correlations between hormonal and nutritional responses, as a means to further characterize the role of hormones in the response of plants to limiting nutrients in soil.

PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 Is a Plant-Specific SEC12-Related Protein That Enables the Endoplasmic Reticulum Exit of a High-Affinity Phosphate Transporter in Arabidopsis

PHOSPHATE TRANSPORTER1 (PHT1) genes encode phosphate (Pi) transporters that play a fundamental role in Pi acquisition and remobilization in plants. Mutation of the Arabidopsis thaliana PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) impairs Pi transport, resulting in the constitutive expression of many Pi starvation–induced genes, increased arsenate resistance, and reduced Pi accumulation. PHF1 expression was detected in all tissues, particularly in roots, flowers, and senescing leaves, and was induced by Pi starvation, thus mimicking the expression patterns of the whole PHT1 gene family. PHF1 was localized in endoplasmic reticulum (ER), and mutation of PHF1 resulted in ER retention and reduced accumulation of the plasma membrane PHT1;1 transporter. By contrast, the PIP2A plasma membrane protein was not mislocalized, and the secretion of Pi starvation–induced RNases was not affected in the mutant. PHF1 encodes a plant-specific protein structurally related to the SEC12 proteins of the early secretory pathway. However, PHF1 lacks most of the conserved residues in SEC12 proteins essential as guanine nucleotide exchange factors. Although it functions in early secretory trafficking, PHF1 likely evolved a novel mechanism accompanying functional specialization on Pi transporters. The identification of PHF1 reveals that plants are also endowed with accessory proteins specific for selected plasma membrane proteins, allowing their exit from the ER, and that these ER exit cofactors may have a phylum-specific origin.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA–insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

A Mutant of the Arabidopsis Phosphate Transporter PHT1;1 Displays Enhanced Arsenic Accumulation

The exceptional toxicity of arsenate [As(V)] is derived from its close chemical similarity to phosphate (Pi), which allows the metalloid to be easily incorporated into plant cells through the high-affinity Pi transport system. In this study, we identified an As(V)-tolerant mutant of Arabidopsis thaliana named pht1;1-3, which harbors a semidominant allele coding for the high-affinity Pi transporter PHT1;1. pht1;1-3 displays a slow rate of As(V) uptake that ultimately enables the mutant to accumulate double the arsenic found in wild-type plants. Overexpression of the mutant protein in wild-type plants provokes phenotypic effects similar to pht1;1-3 with regard to As(V) uptake and accumulation. In addition, gene expression analysis of wild-type and mutant plants revealed that, in Arabidopsis, As(V) represses the activation of genes specifically involved in Pi uptake, while inducing others transcriptionally regulated by As(V), suggesting that converse signaling pathways are involved in plant responses to As(V) and low Pi availability. Furthermore, the repression effect of As(V) on Pi starvation responses may reflect a regulatory mechanism to protect plants from the extreme toxicity of arsenic.