Raquel L. Chan

Homeoboxes in plant development

The homeobox is a 180 bp consensus DNA sequence present in a number of genes involved in developmental processes. This review focuses on the structure and function of plant homeobox genes and of the proteins they encode. Plant homeobox genes have been identified in studies using mutants, degenerate oligonucleotides deduced from conserved sequences, differential screening or binding to known promoters. According to sequence conservation, plant homeoboxes can be subdivided into different families, each comprising several members. Evolutionary studies indicate that the different families have diverged prior to the separation of the branches leading to animals, plants and fungi. Accordingly, members of different families show characteristic structural and functional properties. As an example, kn1-like genes seem to be involved in different aspects of the control of cell fate determination in the shoot meristem; HD-Zip genes, which encode proteins containing a leucine zipper motif adjacent to the homeodomain, are believed to operate at later stages of development; and gl2-like genes are involved in epidermal cell differentiation. Future studies should be oriented to discern the precise function of the many homeobox genes present in plant genomes, and to evaluate their use as modifiers of plant development.

Transcriptional Control of a Plant Stem Cell Niche

Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. Here, we report the genome-wide regulatory potential of WUSCHEL, the key transcription factor for stem cell maintenance in the shoot apical meristem of the reference plant Arabidopsis thaliana. WUSCHEL acts by directly binding to at least two distinct DNA motifs in more than 100 target promoters and preferentially affects the expression of genes with roles in hormone signaling, metabolism, and development. Striking examples are the direct transcriptional repression of CLAVATA1, which is part of a negative feedback regulation of WUSCHEL, and the immediate regulation of transcriptional repressors of the TOPLESS family, which are involved in auxin signaling. Our results shed light on the complex transcriptional programs required for the maintenance of a dynamic and essential stem cell niche.

Hahb-4, a sunflower homeobox-leucine zipper gene, is a developmental regulator and confers drought tolerance to Arabidopsis thaliana plants

Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants. Hahb-4 is a member of Helianthus annuus (sunflower) subfamily I. It is regulated at the transcriptional level by water availability and abscisic acid. In order to establish if this gene plays a functional role in drought responses, transgenic Arabidopsis thaliana plants that overexpress Hahb-4 under the control of the 35S Cauliflower Mosaic Virus promoter were obtained. Transformed plants show a specific phenotype: they develop shorter stems and internodes, rounder leaves and more compact inflorescences than their non-transformed counterparts. Shorter stems and internodes are due to a lower rate in cell elongation rather than to a cell division. Transgenic plants were more tolerant to water stress conditions, showing improved development, a healthier appearance and higher survival rates than wild-type plants. Indeed, either under normal or drought conditions, they produce approximately the same seed weight per plant as wild-type plants under normal growth conditions. Plants transformed with a construct that bears the Hahb-4 promoter fused to gusA show reporter gene expression in defined cell-types and developmental stages and are induced by drought and abscisic acid. Since Hahb-4 is a transcription factor, we propose that it may participate in the regulation of the expression of genes involved in developmental responses of plants to desiccation.

Environmental Regulation of Lateral Root Emergence in Medicago truncatula Requires the HD-Zip I Transcription Factor HB1

Medicago truncatula HB1 is an HD-Zip transcription factor involved in the adaptive developmental response of root architecture to adverse environmental stresses. HB1 reduces lateral root emergence, likely by directly repressing the expression of an auxin-responsive transcription factor from the LBD family, LBD1. The adaptation of root architecture to environmental constraints is a major agricultural trait, notably in legumes, the third main crop worldwide. This root developmental plasticity depends on the formation of lateral roots (LRs) emerging from primary roots. In the model legume Medicago truncatula, the HD-Zip I transcription factor HB1 is expressed in primary and lateral root meristems and induced by salt stress. Constitutive expression of HB1 in M. truncatula roots alters their architecture, whereas hb1 TILLING mutants showed increased lateral root emergence. Electrophoretic mobility shift assay, promoter mutagenesis, and chromatin immunoprecipitation–PCR assays revealed that HB1 directly recognizes a CAATAATTG cis-element present in the promoter of a LOB-like (for Lateral Organ Boundaries) gene, LBD1, transcriptionally regulated by auxin. Expression of these genes in response to abscisic acid and auxin and their behavior in hb1 mutants revealed an HB1-mediated repression of LBD1 acting during LR emergence. M. truncatula HB1 regulates an adaptive developmental response to minimize the root surface exposed to adverse environmental stresses.

Redox Regulation of Plant Homeodomain Transcription Factors

Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells.

Two direct targets of cytokinin signaling regulate symbiotic nodulation in Medicago truncatula.

In legume plants, cytokinins are necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. Biochemical and reverse genetic approaches identified two transcription factors from the GRAS (NSP2) and bHLH families as direct targets of cytokinin signaling pathways in legume roots. These transcription factors act at the convergence of phytohormonal and nodulation symbiotic cues. Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.

A MONOMER-DIMER EQUILIBRIUM MODULATES THE INTERACTION OF THE SUNFLOWER HOMEODOMAIN LEUCINE-ZIPPER PROTEIN HAHB-4 WITH DNA

We have analysed the interaction of the sunflower homeodomain leucine-zipper (Hd-Zip) protein Hahb-4 with DNA. The complete Hd-Zip domain from Hahb-4 was able to select specific sequences from a random oligonucleotide mixture that contained a 9-bp core with four fixed and five degenerate positions. Analysis of the binding of some of the selected sequences suggests that Hahb-4 preferentially binds the dyad-symmetrical sequence CAAT(A/T)ATTG. Single-nucleotide replacements at positions 1, 5 or 9 of this sequence produced a decrease in binding of 2-4-fold. DNA binding as a function of protein concentration was non-hyperbolic. This behaviour could be explained by an equation in which dimer formation is a pre-requisite for DNA binding. A global dissociation constant (Kd) of 1.31x10(-14) M2 could be calculated. The removal of the leucine zipper promoted a change in specificity and a decrease in binding affinity (Kd=5. 03x10(-5) M). Mutation of Phe-20 of the homeodomain into Leu completely abolished DNA binding. The mutant protein, however, was able to inhibit DNA binding by the non-mutant form, presumably through the formation of heterodimers. The analysis of this inhibitory effect at different mutant concentrations allowed the estimation of the Kd for the dimer-monomer equilibrium [about (2-4)x10(-6) M]; from this, a Kd of 3-6x10(-9) M for the dimer-DNA complex could be estimated. The results obtained indicate that the formation of dimers is the main factor influencing the interaction of Hahb-4 with DNA. It is proposed that shifts in a dimer-monomer equilibrium could be used within the cell to modulate the interaction of this protein with target genes.

HAHB4, a sunflower HD-Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD-Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl-jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense-related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4-silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense-related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA-isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation.

Transient transformation of sunflower leaf discs via an Agrobacterium -mediated method: applications for gene expression and silencing studies

The sunflower belongs to the Compositae family and is an economically important crop because of the quality of its oil. Unfortunately, molecular analyses are limited due to the lack of genomic information, mutant libraries and efficient and rapid transformation protocols. In a wide variety of species, Agrobacterium-mediated transient transformation is a useful tool that can provide valuable insight into many biological processes. However, this technology has not been routinely applied to the sunflower because of difficulties with infiltration. Here, we present an optimized protocol for Agrobacterium–mediated transient transformation of leaf discs. Using this procedure, we were able to quickly overexpress or silence a given gene, enabling us to study several biochemical processes and characterize sunflower regulatory sequences. One of the major advantages of this approach is that in only 1 work-week it is possible to acquire considerable molecular information while avoiding the use of controversial heterologous systems. Transforming heterologous species is frequently unacceptable, as the conservation of molecular events in many cases is not well documented.

Role of recently evolved miRNA regulation of sunflower HaWRKY6 in response to temperature damage

MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated. Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing. Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature. Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress.