Transcription factors (TFs) are DNA-binding proteins that interact with other transcriptional regulators, including chromatin remodeling/modifying proteins, to recruit or block access of RNA polymerases to the DNA template Plant genomes devote approximately 7% of their coding sequence to TFs, which

Legume transcription factors: global regulators of plant development and response to the environment

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. 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. 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.

/pdf/a-community-resource-for-high-throughput-quantitative-rt-pcr-2t5tnmbto6.pdf

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

The symbiosis between legume plants and rhizobia causes the development of new organs, nodules which function as an apparatus for nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter GH3 fused to β-glucuronidase (GUS) was analyzed in L. japonicus roots, and showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, generation of GUS signal was observed at the dividing outer cortical cells during the first nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting distinct auxin involvement in the determinate nodule development. Numbers and the development of nodules were affected by auxin transport inhibitors (1-naphthylphthalamic acid, NPA and triindobenzoic acid, TIBA), and by a newly synthesized auxin antagonist, α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA). The common phenotypical alteration by these auxin inhibitors was the inhibition in forming lenticel which is normally developed on the nodule surface from the root outer cortex. The inhibition of lenticel formation was correlated with the inhibition of nodule vascular bundle development. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner.

/pdf/involvement-of-auxin-distribution-in-root-nodule-development-2ucxsdskiw.pdf

Involvement of auxin distribution in root nodule development of Lotus japonicus

Nodules in the family Leguminosae come in many different shapes and forms, depending partly on the host that bears them. This review focuses on the peripheral tissues that surround the infected tissues of four anatomically distinct classes of nodules: nodules with indeterminate meristems (from the Trifolieae and Fabeae tribes); desmodioid nodules with determinate meristems (from the Phaseoleae and Loteae tribes); aeschynomenoid nodules (Arachis hypogaea L., peanut); and lupinoid nodules (Lupinus sp. L., lupine). I have especially stressed the importance of the three-dimensionality of the organ, because not all nodules display a radial symmetry as it is often assumed. The goal of this review is to provide a strong base of nodule structure so that forthcoming molecular studies can integrate this information into their approach.

Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types

During the past decade, the legume Lotus japonicus has emerged as an important model system for study of symbiotic nitrogen fixation. Controlled expression of genes involved in symbiosis from an inducible promoter at specific time points would be a valuable tool for investigating gene function in L. japonicus. We have attempted to study the function of the putative transcription factors LjNDX and LjCPP1 by expression from the GVG inducible system. This study showed that the GVG system itself causes growth disturbances in L. japonicus. Shoot internode elongation and root pericycle cell division are affected when the chimeric GVG transcription factor is activated. We suggest that deficient auxin signaling could cause the phenotype observed and conclude that the GVG inducible system is not well suited for use in the model legume L. japonicus.

The glucocorticoid-inducible GVG system causes severe growth defects in both root and shoot of the model legume Lotus japonicus.

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndxgenes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

The Lotus japonicus ndx gene family is involved in nodule function and maintenance.

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The Lotus japonicus ndx gene family is involved in nodule function and maintenance.