Understanding the Expression Dynamics of Rhizobial Nifh and Dry Bean NR and GS Genes.

Carbon and Nitrogen (N) assimilation in plants found to be interacting, playing regulatory role in gene expression and plant morphological development. Symbiotic bacteria (Rhizobium) associated with roots of legume crops greatly augment fixation of N to the plant system through biological nitrogen fixation (BNF). Bacterial metalloenzyme ‘nitrogenase’ act as BNF catalyst and involve two components, a heterotetrameric core, and dinitrogenase reductase encoded by nifH gene, a biological marker for BNF. Thus, BNF involve nifH gene expression in root nodules, increasing plant efficiency to utilize available N from soil. In our field study during summer 2016, we found that dry bean (Phaseolus vulgaris L.) cultivars fix variable amount of N from soil. Based on field data, we hypothesize that efficiency of symbiotic relationship and effective nodule production in dry bean plants resulting in BNF should correlate with its nitrogen assimilation, and act as one factor in rank classification of dry bean cultivars. Cultivars with higher nifH gene expression should fix more N from atmosphere. Consequently, expressions of plant N-assimilatory genes, nitrate reductase (NR) and glutamine synthetase (GS) genes, should also positively correlate with the nifH gene expression at given time points. A negative correlation will be more interesting possibly indicating time lag between the fixation and utilization of N, depending on critical growth stages of plant when more N is needed. Two different plant growth stage were chosen, V₃ (third trifoliate stage) and R₁ (first flowering) stages to test the hypothesis, and leaf and nodule samples were collected. We optimized the quantity of required sample for efficient total RNA recovery from leaf and nodules. Total RNA from root nodules and leaves of four pinto bean cultivars were extracted and reverse transcribed using random hexamer primers to make cDNA, utilizable for both plant and bacterial gene expression study. We have designed, and validated PCR (polymerase chain reaction) primers to study nifH, NR and GS gene expressions along with housekeeping reference genes, actin for dry bean and recA for bacteria. q-RT PCR (quantitative reverse transcription PCR) will be performed to study gene expressions using SYBR Green fluorescence dye. BNF will be estimated using stable isotope (¹⁵N) dilution technique.

Our study will provide molecular evidence for differential nifH gene expression for different cultivars fixing variable amount of N, and associated changes in NR and GS gene expression levels at the two growth stages. This outcome will strengthen the existing understanding of nitrogen need, assimilation and fixation by plants, and their regulatory relationships affecting the plant growth.