Genome-Wide Differential DNA Methylation and miRNA Expression Profiling Reveals Epigenetic Regulatory Mechanisms Underlying Nitrogen-Limitation-Triggered Adaptation and Use Efficiency Enhancement in Allotetraploid Rapeseed

Abstract

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

miR160: An Indispensable Regulator in Plant

“…In the field, macronutrients and micronutrients are necessary for plants. As a nitrogen-starvation responsive gene, miR160 regulated the establishment of root system architecture through the auxin signaling pathway under nitrogen starvation conditions in plants ( Liang et al, 2012 ; Hua et al, 2020 ; Singh and Singh, 2021 ). miR160 could enhance LRs or ARs developments under N-deficiency conditions to maximize the capacity of many plants, such as Arabidopsis , cotton, and B. napus , to uptake the little available nitrogen ( Magwanga et al, 2019 ).…”

Noncoding-RNA-Mediated Regulation in Response to Macronutrient Stress in Plants

“…Global screening of miRNAs responsive to N starvation was first reported in Arabidopsis, in which miR160, miR780, miR826, miR842, and miR846 exhibited increased expression, while miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 showed decreased expression upon N stress [ 57 ]. Likewise, comparative expression profiling by deep sequencing helped to identified N-responsive miRNAs from shoots and roots of 7-day N-starved rice [ 50 ], from leaves and roots of two wheat cultivars subjected to chronic or short-term N stress [ 109 , 110 ], and from shoots and roots of rapeseeds with 0 or 72 h of N-limitation treatment [ 111 ].…”

“…For instance, 13 miRNAs showed similar expression changes in roots and shoots of soybeans under P deficiency, while 6 miRNAs had opposite expression changes in these two tissues [ 114 ]. In rapeseed, 11 upregulated and 15 downregulated miRNAs were specifically identified in roots under N starvation, whereas 25 upregulated and 23 downregulated miRNAs were specifically identified in shoots [ 111 ].…”

“…In the same study, the confirmation of MADS25 as a novel target gene of osa-miR444a.4-3p, which was also identified as a N-starvation-responsive miRNA by sRNA-Seq, was aided by analyzing the degradome sequencing data of the N-starved rice [ 50 ]. Similarly, combined miRNAome and degradome analysis also helped to reveal the involvement of the miR827-NLA pathway in limited N-induced leaf senescence, as well as the involvement of the miR171-SCL6 and miR160-ARF17 pathways in roots grown under N deprivation [ 111 ].…”

Triticale doubled haploid plant regeneration factors linked by structural equation modeling

“…In plants, DNA-methylated cytosine appears in two types of sequence contexts: symmetric (CG, CHG) and asymmetric (CHH) (H is A, C, or T) (Cokus et al 2008 ; Lister et al 2008 ). The distribution of methylated cytosine varies across sequence contexts (Feng et al 2010 ) and genome regions (Hua et al 2020 ) and fluctuates during biological processes (Li et al 2019 ). Similarly, there is variability regarding methylation in each context depending on the tissue studied, so in the rapeseed genome, the most significant differences were identified between shoots and roots in the context of CHH (Hua et al 2020 ).…”