Abstract
Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter beta-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.
Highlights
Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil
Nodulation involves the intimate relationship between soil bacteria and legume plants, which results in the formation of a novel organ, the nodule, in which the bacteria reside and provide a steady source of nitrogen to the plant
This study clearly demonstrated that the use of such quantitative reverse transcription (qRT)-PCR primer libraries provides 100- to 1,000-fold greater sensitivity in accurately measuring transcription factors (TFs) gene expression compared with DNA microarray technologies
Summary
Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. Such results suggest that TFs involved in nodulation may be identified by directly analyzing their transcriptional response to inoculation Based on such facts, large-scale DNA microarray studies identified a variety of putative TF genes that were differentially expressed during nodule initiation and development (El Yahyaoui et al, 2004; Kouchi et al, 2004; Benedito et al, 2008; Brechenmacher et al, 2008). This study clearly demonstrated that the use of such qRT-PCR primer libraries provides 100- to 1,000-fold greater sensitivity in accurately measuring TF gene expression compared with DNA microarray technologies Since this initial paper, other primer libraries have been developed for the various putative regulatory genes in Oryza sativa (Caldana et al, 2007) and Medicago truncatula (Kakar et al, 2008). These libraries, as well as the original Arabidopsis library, were used in large-scale analyses to identify putative regulatory genes that are differentially expressed in different plant tissues, in response to pathogens and microbial elicitors, and after treatment with nitrogen and phosphorous (Czechowski et al, 2004; Scheible et al, 2004; McGrath et al, 2005; Caldana et al, 2007; Libault et al, 2007; Morcuende et al, 2007; Kakar et al, 2008; Verdier et al, 2008; Gruber et al, 2009)
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