We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant.

In plants, metal transporters are responsible for metal uptake, translocation and homeostasis. These metals include essential nutrients such as zinc (Zn) and manganese (Mn) or non-essential metals like cadmium (Cd) and lead (Pb). Although a few metal transporters have been well characterized in model plants, little is known about their functionality in rapeseed (Brassica napus). In the study, 22 NRAMP transporter genes from B. napus genome were identified and annotated using bioinformatics and high-throughput RNA-sequencing (RNA-seq). Based on the sequence identity, these NRAMP transporters can be classified into 6 subfamilies. RNA-seq analysis revealed that 19 NRAMP transporters were detected and some of the genes were well confirmed by qRT-PCR. Ten NRAMP transporters (45.5%, 10/22) were found to be differentially expressed (> 2 fold change, p < 0.05) under Cd exposure. As an example, we specified expression of BnNRAMP1b under Cd exposure. BnNRAMP1b is a constitutive gene expressing throughout all development stages including seedlings, vegetative tissue, flowers and siliques. Expression of BnNRAMP1b can be strongly induced in seedlings exposed to 80, 160 and 240 μM Cd. To define whether BnNRAMP1b was specific for Cd transport, a yeast (wild-type, BY4741) system with its mutants (ycf1, zrc1, and smf1) defective in transport activity of Cd, Zn and Mn, respectively were tested. Compared to empty vectors (pYES2), cells carrying BnNRAMP1b can rescue the transport functions. As a consequence, excess Cd, Zn and Mn were taken in the cells, which led to metal toxicity, suggesting that BnNRAMP1b is responsible for transport of these metals in B. napus. Using our previously created degradome datasets, we found that BnNRAMP1b could be cleaved by miR167, suggesting that BnNRAMP1b is a target of miR167 in B. napus. The contrasting expression pattern of BnNRAMP1b and miR167 under Cd stress supported the post-transcriptional regulation of BnNRAMP1b by miR167.

There is increasing evidence of long non-coding RNA (lncRNA) involvement in a variety of biological responses to environmental stresses.