Genome-scale characterization of the vacuole nitrate transporter Chloride Channel (CLC) genes and their transcriptional responses to diverse nutrient stresses in allotetraploid rapeseed.

Qiong Liao,Chun-Yun Guan,Hai-Xing Song,Ying-Peng Hua,Zhen-Hua Zhang,Ting Zhou,Qing-Fen Han,Jun-Yue Yao,Anil Kumar Singh

doi:10.1371/journal.pone.0208648

Abstract

The Chloride Channel (CLC) gene family is reported to be involved in vacuolar nitrate (NO3-) transport. Nitrate distribution to the cytoplasm is beneficial for enhancing NO3- assimilation and plays an important role in the regulation of nitrogen (N) use efficiency (NUE). In this study, genomic information, high-throughput transcriptional profiles, and gene co-expression analysis were integrated to identify the CLCs (BnaCLCs) in Brassica napus. The decreased NO3- concentration in the clca-2 mutant up-regulated the activities of nitrate reductase and glutamine synthetase, contributing to increase N assimilation and higher NUE in Arabidopsis thaliana. The genome-wide identification of 22BnaCLC genes experienced strong purifying selection. Segmental duplication was the major driving force in the expansion of the BnaCLC gene family. The most abundant cis-acting regulatory elements in the gene promoters, including DNA-binding One Zinc Finger, W-box, MYB, and GATA-box, might be involved in the transcriptional regulation of BnaCLCs expression. High-throughput transcriptional profiles and quantitative real-time PCR results showed that BnaCLCs responded differentially to distinct NO3- regimes. Transcriptomics-assisted gene co-expression network analysis identified BnaA7.CLCa-3 as the core member of the BnaCLC family, and this gene might play a central role in vacuolar NO3- transport in crops. The BnaCLC members also showed distinct expression patterns under phosphate depletion and cadmium toxicity. Taken together, our results provide comprehensive insights into the vacuolar BnaCLCs and establish baseline information for future studies on BnaCLCs-mediated vacuolar NO3- storage and its effect on NUE.

Highlights

Nitrogen (N) is a fundamental non-mineral macronutrient, which is essential for the growth and development of higher plants [1]
We found that genome-wide duplication and segmental duplication of the chloride channel (CLC) genes contributed to a relatively large CLC gene family in B. napus
We found that the clca-2 mutant showed enhanced N assimilation ability and high N use efficiency (NUE)

Summary

Introduction

Nitrogen (N) is a fundamental non-mineral macronutrient, which is essential for the growth and development of higher plants [1]. China is the largest N-consuming country worldwide; massive amounts of N are applied annually as fertilizer, crop yields are declining in some areas [2,3]. Enhancing plant N use efficiency (NUE) is critical for developing sustainable agriculture [4]. Oilseed rape (Brassica napus L.) is a staple oil crop and has a high N requirement. To maintain its optimum yield, relatively high amounts of N fertilizer (from 150 to 300 kg N hm-2) are applied to soils [5,6], but only 30–50% of the applied N fertilizer is taken up from soil by crops [7]. The development of N-efficient cultivars through genetic improvement of crop NUE is a cost-effective and environmental friendly way to reduce excessive N in soils [8]. Four NO3- transporters, namely nitrate transporter 1 (NRT1), nitrate transporter 2 (NRT2), chloride channel (CLC)

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