Elsevier

Gene

Volume 546, Issue 2, 10 August 2014, Pages 327-335
Gene

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.)

https://doi.org/10.1016/j.gene.2014.05.057Get rights and content

Highlights

  • Isolated EcDof2 (GenBank-KF261117) showed max. homology with Dof2 of Zea mays.

  • EcDof2 expression was higher in roots as compared to shoot in GE1437 and GE3885.

  • Light and N influenced EcDof expressions suggest complex regulation of C/N genes.

  • Expressions of genes of C/N metabolism are affected by the ratio of EcDof1/Dof2.

  • EcDof1/Dof2 ratio serves as index for measuring N-responsiveness and NUE of crops.

Abstract

In order to gain insights into the mechanism of high nitrogen use efficiency (NUE) of finger millet (FM) the role of Dof2 transcription factor (TF), which is a repressor of genes involved in C/N metabolism was investigated. The partial cDNA fragment of EcDof2 (912-bp; GenBank acc. no. KF261117) was isolated and characterized from finger millet (FM) that showed 63% and 58% homology with Dof2 of Zea mays at nucleotide and protein level, respectively. Its expression studies were carried out along with the activator EcDof1 in two genotypes (GE3885, high protein genotype (HPG); GE1437, low protein genotype (LPG)) of FM differing in grain protein contents (13.8% and 6.2%) showed that EcDof2 is expressed in both shoot and root tissues with significantly (p  0.05) higher expression in the roots. The diurnal expression of both EcDof1 and EcDof2 in shoots was differential having different time of peak expression indicating a differential response to diurnal condition. Under continuous dark conditions, expression of EcDof1 and EcDof2 oscillated in both the genotypes whereas on illumination, the fold expression of EcDof1 was higher as compared to EcDof2. Under increasing nitrate concentration, EcDof2 expression increases in roots and shoots of LPG while it remains unchanged in HPG. However, the EcDof1 expression was found to increase in both genotypes. Further, time kinetics studies under single nitrate concentration revealed that EcDof2 was repressed in the roots of both genotypes whereas EcDof1 oscillated with time. The EcDof1/EcDof2 ratio measured showed differential response under different light and nitrogen conditions. It was higher in the roots of HPG indicating higher activation of genes involved in N uptake and assimilation resulting in high grain protein accumulation. The results indicate that both light and nitrogen concentration influence Dof1 and Dof2 expression and suggests a complex pattern of regulation of genes influenced by these plant specific TFs. In nutshell, the Dof1/Dof2 ratio can serve as an index for measuring the N responsiveness and NUE of crops and can be further validated by Dof2 knock down approach.

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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