Fluctuation of Dof1/Dof2 expression ratio under the influence of varying nitrogen and light conditions: involvement in differential regulation of nitrogen metabolism in two genotypes of finger millet (Eleusine coracana L.)
Introduction
Worldwide, the nitrogen use efficiency (NUE) for crop plants is of great concern but due to lack of knowledge about precise regulatory mechanisms to explain NUE in crop plants hampers the goal of agricultural productivity. Therefore, the identification of potential candidate genes/proteins in the regulation of NUE will serve as biomarker(s) for determining genotypic potential for optimization of nitrogen input in agriculture (Kant et al., 2011, Kumar et al., 2009). The Dof (DNA binding with one finger) factors represent a plant-specific family of transcription factors (TFs) that appear to be a key regulator in the accumulation of grain protein and yield attribute through regulation of key enzymes of C/N metabolism involved in source to sink relationship during grain filling stage (Gupta et al., 2011, Gupta et al., 2012). Dof proteins recognize specifically the AAAG core motif (Plesch et al., 2001) occurring in different promoter regions, and have been implicated in the regulation of various genes including tissue-specifically expressed, light-regulated and stress/phytohormone-responsive genes (Ward et al., 2005, Yanagisawa, 2000, Yanagisawa and Sheen, 1998). Specific members of the Dof TF gene family has been found to act like master regulators by simultaneously regulating the genes involved in carbon (PEPC, PK, CyPPDK) and nitrogen (NR, GS, GOGAT etc.) metabolism (Gupta et al., 2013, Kumar et al., 2009, Yanagisawa, 2000).
This important finding led to the generation of transgenic lines carrying over expressed maize Dof1 that showed improved NUE under low nitrogen conditions (Wang et al., 2013, Yanagisawa et al., 2004). Furthermore, maize Dof1 is constitutively expressed in leaves, stems and roots, whereas the Dof2 which is structurally closest to Dof1 is expressed mainly in stems and roots. Dof2 binds to DNA with identical or very similar sequence specificity to Dof1. Co-transfection experiments with expression vectors for Dof proteins and their putative target promoters fused to reporter genes have provided direct evidence that Dof proteins function as transcriptional activators or repressors through direct interaction with DNA. Dof1 differently elevated the promoter activity in etiolated and greening protoplasts, suggesting involvement of Dof1 in the light-regulated gene expression (Yanagisawa, 2000, Yanagisawa and Sheen, 1998). On the other hand, maize Dof2 having the closest relationship with Dof1 blocked trans-activation by Dof1 and repressed activity of the C4-type PEPC promoter (Yanagisawa and Sheen, 1998) suggesting that these Dof proteins with opposite activities might be involved in the control of expression of some genes. The results clarified the transcriptional activator and repressor activity of Dof domain proteins in which maize Dof2 showed both activator and repressor activities on different promoters (Yanagisawa, 2000). Therefore, to define the role of Dof2 in context to Dof1 regulation under varying nitrogen and light conditions is the most important strategy for development of high NUE crops.
The burgeoning world population increases the demand of food which results in the overuse of inorganic nitrogenous fertilizers that are hazardous to the environment. However, to minimize the environmental pollution and for fulfilling the high global demand of organic produce, it requires the low nitrogen responsive genotypes with greater NUE and grain yields. Since, common cereal genomes have a limited gene pool for this trait (high NUE) (Hilu et al., 1979), research on understanding the mechanism of the high NUE of finger millet (FM) would seem appropriate. It thrives on almost no nitrogen input, has a high NUE and accumulates high amounts of proteins in its grains. Also, it appears that the FM crop has devised unique regulatory controls to achieve high NUE under limiting nitrogen conditions. Furthermore, to understand the mechanisms of high NUE in FM, we earlier isolated and cloned EcDof1 gene and investigated its role in FM genotypes (Gupta et al., 2011, Gupta et al., 2012, Gupta et al., 2013, Kumar et al., 2009). Since, circadian regulated transcription factors induce or repress the expression of genes involved in C/N metabolism and nitrogen assimilation (Foyer and Noctor, 2002, Kumar et al., 2013, Stitt et al., 2002) is integrated with photosynthesis therefore, the expression of EcDof1 and EcDof2 was studied under different light–dark and nitrogen conditions in two genotypes. The present study was aimed to isolate, characterize and understand the role of EcDof2 expression under different light–dark and nitrogen conditions in FM. Also, the ratio of EcDof1/EcDof2 was investigated in order to understand whether the ratio plays any significant role in grain protein accumulation and yield vis-a-vis high NUE in two FM genotypes differing in grain protein content.
Section snippets
Plant material and growth conditions
The seeds of finger millet (FM) genotypes were collected from CRC, Pantnagar. In the present study, two genotypes of FM namely, GE3885 (HPG) and GE1437 (LPG) that have contrasting grain protein (13.8% and 6.2%) but nearly similar carbohydrate contents (64.8% and 75.6%), respectively, were used for all the experiments. Seeds of the genotypes HPG and LPG of FM were sterilized with Tween-20 for 10 min followed by 0.1% HgCl2 treatment for 5 min and thoroughly rinsed (3–5 times) in sterile double
Results and discussion
The Dof2 has not been reported so far in FM, therefore the present study was undertaken to isolate, characterize and understand the role of EcDof2 expression under different light and nitrogen conditions. Further, to investigate the molecular basis of light and nitrogen responsiveness, we have also studied the ratio of Dof1/Dof2 expression that might play significant role in grain protein accumulation and yield vis-a-vis differential C and N metabolism in two FM genotypes differing in grain
Conflict of interest
There is no Conflict of Interest.
Acknowledgments
The financial assistance provided to SG under WOS-A scheme by the Department of Science and Technology (DST SR/WOS-A/LS-125/2011 (G) dated 17th feb 2012), Government of India, is duly acknowledged. The logistic support provided by the Director, Experiment Station, G.B. Pant University of Agriculture and Technology, Pantnagar is also thankfully acknowledged.
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First two authors contributed equally.