A PLETHORA-auxin transcription module controls cell division plane rotation through MAP65 and CLASP.

Despite their pivotal role in plant development, control mechanisms for oriented cell divisions have remained elusive. Here, we describe how a precisely regulated cell division orientation switch in an Arabidopsis stem cell is controlled by upstream patterning factors. We show that the stem cell regulatory PLETHORA transcription factors induce division plane reorientation by local activation of auxin signaling, culminating in enhanced expression of the microtubule-associated MAP65 proteins. MAP65 upregulation is sufficient to reorient the cortical microtubular array through a CLASP microtubule-cell cortex interaction mediator-dependent mechanism. CLASP differentially localizes to cell faces in a microtubule- and MAP65-dependent manner. Computational simulations clarify how precise 90° switches in cell division planes can follow self-organizing properties of the microtubule array in combination with biases in CLASP localization. Our work demonstrates how transcription factor-mediated processes regulate the cellular machinery to control orientation of formative cell divisions in plants.

CLASP-mediated cortical microtubule organization guides PIN polarization axis.

Recent evidence indicates a correlation between orientation of the plant cortical microtubule cytoskeleton and localization of polar cargoes. However, the molecules and mechanisms that create this correlation have remained unknown. Here we show that, in Arabidopsis thaliana, the microtubule orientation regulators CLASP and MAP65 (refs 3, 4) control the abundance of polarity regulator PINOID kinase at the plasma membrane. By localized upregulation of clathrin-dependent endocytosis at cortical microtubule- and clathrin-rich domains orthogonal to the axis of polarity, PINOID accelerates the removal of auxin transporter PIN proteins from those sites. This mechanism links directional microtubule organization to the polar localization of auxin transporter PIN proteins, and clarifies how microtubule-enriched cell sides are kept distinct from polar delivery domains. Our results identify the molecular machinery that connects microtubule organization to the regulation of the axis of PIN polarization.

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors.

BACKGROUND: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. RESULTS: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. CONCLUSION: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.

Identification of potential early regulators of aphid resistance in Medicago truncatula via transcription factor expression profiling.

*Resistance to aphids has been identified in a number of plant species, yet the molecular mechanisms underlying aphid resistance remain largely unknown. *Using high-throughput quantitative real-time PCR technology, the transcription profiles of 752 putative Medicago truncatula transcription factor genes were analysed in a pair of susceptible and resistant closely related lines of M. truncatula following 6 and 12 h of bluegreen aphid (Acyrthosiphon kondoi) infestation. *Eighty-two transcription factor genes belonging to 30 transcription factor families were responsive to bluegreen aphid infestation. More transcription factor genes were responsive in the resistant interaction than in the susceptible interaction; of the 36 genes that were induced at 6 and/or 12 h, 32 were induced only in the resistant interaction. Bluegreen aphid-induced expression of a subset of these genes was correlated with the presence of AKR, a single dominant gene conferring resistance to bluegreen aphids. Similar transcription factor expression patterns of this subset were associated with bluegreen aphid resistance in other M. truncatula genetic backgrounds, as well as with resistance to pea aphid (Acyrthosiphon pisum). *Our results suggest that these transcription factors are among the early aphid-responsive genes in resistant plants, and may play important roles in resistance to multiple aphid species.

A gene expression atlas of the model legume Medicago truncatula.

Legumes played central roles in the development of agriculture and civilization, and today account for approximately one-third of the world's primary crop production. Unfortunately, most cultivated legumes are poor model systems for genomic research. Therefore, Medicago truncatula, which has a relatively small diploid genome, has been adopted as a model species for legume genomics. To enhance its value as a model, we have generated a gene expression atlas that provides a global view of gene expression in all major organ systems of this species, with special emphasis on nodule and seed development. The atlas reveals massive differences in gene expression between organs that are accompanied by changes in the expression of key regulatory genes, such as transcription factor genes, which presumably orchestrate genetic reprogramming during development and differentiation. Interestingly, many legume-specific genes are preferentially expressed in nitrogen-fixing nodules, indicating that evolution endowed them with special roles in this unique and important organ. Comparative transcriptome analysis of Medicago versus Arabidopsis revealed significant divergence in developmental expression profiles of orthologous genes, which indicates that phylogenetic analysis alone is insufficient to predict the function of orthologs in different species. The data presented here represent an unparalleled resource for legume functional genomics, which will accelerate discoveries in legume biology.

Identification of transcription factors involved in root apex responses to salt stress in Medicago truncatula.

The root apex contains meristematic cells that determine root growth and architecture in the soil. Specific transcription factor (TF) genes in this region may integrate endogenous signals and external cues to achieve this. Early changes in transcriptional responses involving TF genes after a salt stress in Medicago truncatula (Mt) roots were analysed using two complementary transcriptomic approaches. Forty-six salt-regulated TF genes were identified using massive quantitative real-time RT-PCR TF profiling in whole roots. In parallel, Mt16K+ microarray analysis revealed 824 genes (including 84 TF sequences) showing significant changes (p < 0.001) in their expression in root apexes after a salt stress. Analysis of salt-stress regulation in root apexes versus whole roots showed that several TF genes have more than 30-fold expression differences including specific members of AP2/EREBP, HD-ZIP, and MYB TF families. Several salt-induced TF genes also respond to other abiotic stresses as osmotic stress, cold and heat, suggesting that they participate in a general stress response. Our work suggests that spatial differences of TF gene regulation by environmental stresses in various root regions may be crucial for the adaptation of their growth to specific soil environments.

Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling.

Legume seeds represent a major source of proteins for human and livestock diets. The model legume Medicago truncatula is characterized by a process of seed development very similar to that of other legumes, involving the interplay of sets of transcription factors (TFs). Here, we report the first expression profiling of over 700 M. truncatula genes encoding putative TFs throughout seven stages of seed development, obtained using real-time quantitative RT-PCR. A total of 169 TFs were selected which were expressed at late embryogenesis, seed filling or desiccation. The site of expression within the seed was examined for 41 highly expressed transcription factors out of the 169. To identify possible target genes for these TFs, the data were combined with a microarray-derived transcriptome dataset. This study identified 17 TFs preferentially expressed in individual seed tissues and 135 corresponding co-expressed genes, including possible targets. Certain of the TFs co-expressed with storage protein mRNAs correspond to those already known to regulate seed storage protein synthesis in Arabidopsis, whereas the timing of expression of others may be more specifically related to the delayed expression of the legumin-class storage proteins observed in legumes.

A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula.

BACKGROUND: Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs), which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. RESULTS: We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. CONCLUSION: High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

Systemic signaling of the plant nitrogen status triggers specific transcriptome responses depending on the nitrogen source in Medicago truncatula.

Legumes can acquire nitrogen (N) from NO(3)(-), NH(4)(+), and N(2) (through symbiosis with Rhizobium bacteria); however, the mechanisms by which uptake and assimilation of these N forms are coordinately regulated to match the N demand of the plant are currently unknown. Here, we find by use of the split-root approach in Medicago truncatula plants that NO(3)(-) uptake, NH(4)(+) uptake, and N(2) fixation are under general control by systemic signaling of plant N status. Indeed, irrespective of the nature of the N source, N acquisition by one side of the root system is repressed by high N supply to the other side. Transcriptome analysis facilitated the identification of over 3,000 genes that were regulated by systemic signaling of the plant N status. However, detailed scrutiny of the data revealed that the observation of differential gene expression was highly dependent on the N source. Localized N starvation results, in the unstarved roots of the same plant, in a strong compensatory up-regulation of NO(3)(-) uptake but not of either NH(4)(+) uptake or N(2) fixation. This indicates that the three N acquisition pathways do not always respond similarly to a change in plant N status. When taken together, these data indicate that although systemic signals of N status control root N acquisition, the regulatory gene networks targeted by these signals, as well as the functional response of the N acquisition systems, are predominantly determined by the nature of the N source.