Real-time kinetics of cadmium transport and transcriptomic analysis in low cadmium accumulator Miscanthus sacchariflorus

Abstract

The molecular mechanism of low Cd influxes and accumulation in Miscanthus sacchariflorus is revealed by RNA sequencing technique. Soil cadmium (Cd) pollution has posed a serious threat to our soil quality and food security as well as to human health. Some wild plants exhibit high tolerance to heavy metals stress. However, mechanisms of Cd tolerance of wild plants remain to be fully clarified. In this study, we found that two Miscanthus species, Miscanthus (M.) sacchariflorus and M. floridulus, showed different Cd-tolerant mechanisms. M. sacchariflorus accumulated less Cd in both root and leaf by limiting Cd uptake from root and showed superior Cd tolerance, while M. floridulus not only absorbs more Cd from root but also transports more Cd to shoot. To investigate the molecular mechanism of different Cd uptake patterns in the two Miscanthus species, we analyzed the transcriptome of M. sacchariflorus and identified transcriptional changes in response to Cd in roots by high-throughput RNA-sequencing technology. A total of 92,985 unigenes were obtained from M. sacchariflorus root cDNA samples. Based on the assembled de novo transcriptome, 681 DEGs which included 345 upregulated and 336 downregulated genes were detected between two libraries of untreated and Cd-treated roots. Gene ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Cd stress are predominately involved in metabolic pathway, starch and sucrose and biosynthesis of secondary metabolites and metal ion transporters. Quantitative RT-PCR was employed to compare the expression levels of some metal transport genes in roots of two Miscanthus species, and the genes involved in Cd uptake from root and transfer from root to shoot were extremely different. The results not only enrich genomic resource but also help to better understand the molecular mechanisms of Cd accumulation and tolerance in wild plants.

Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium

“…To explain molecular mechanisms of superior Cd tolerance in M . sacchariflorus , we analyzed our transcriptomic data (Guo et al ) from roots exposed to 100 µ M Cd solutions for 24 h. We found that two malate dehydrogenase ( MsMDH1 and MsMDH2 ) and Al‐activated malate transporter 1 ( MsALMT1 ) genes were upregulated under Cd stress. MsMDH1 was identified as a plastidial NAD‐dependent malate dehydrogenase ( MspdMDH ) (Fig.…”

“…cDNA was synthesized from 1 µg of total RNA using reverse transcriptase PrimeScript RT Master Mix (DRR036; Takara, Tokyo, Japan) with the following conditions: 15 min at 37°C and 5 s at 85°C. qRT‐PCR was performed in a LightCycler 480 (Roche, Sussex, UK) using SYBR Primix Ex Taq kit (DRR420; Takara, Tokyo, Japan) according to the method described previously (Bustin et al , Guo et al ). Briefly, a total of 10 µl reaction solutions including 1 µl of 10‐fold‐dilution cDNA, 5 µl of SYBR Primix Ex Taq and 0.4 µ M forward and reverse primers were performed with the LightCycler 480 machine with the following cycling conditions: 30 s at 95°C, 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 10 s. Primer pairs for genes were designed with primer premier 5.0 software.…”

Unveiling the transcriptomic complexity of Miscanthus sinensis using a combination of PacBio long read- and Illumina short read sequencing platforms

“…The whole genome sequence of Miscanthus is not yet available, but access to functional genomic resources of this species are required to understand the molecular processes underlying their suitability for bioenergetics applications. With the development of new sequencing techniques, transcriptome analysis has been proven to be a very powerful means of gene discovery, genome annotation, and deep exploration of genes that contribute to phenotypic traits [9][10][11]. A few NGS RNA-Seq transcriptome databases of M. sinensis, M. sacchari orus, and M. lutarioriparius have been reported previously [12,13,38], but these studies were limited by either transcript length and/or the number of transcript isoforms.…”

“…For example, the key C4 photosynthesis genes, such as phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) were obviously identi ed in the 'primary metabolism' category with more than 10 alternative splicing isoforms (Additional le 4). PEPC and PPDK are two important enzymes catalyse the early steps in the photosynthetic assimilation of CO 2 in C4 plants [11]. The regulation of the PEPC and PPDK alternative spliced isoforms may be directly related to the photosynthesis e ciency of plants.…”

“…For example, extensive transcriptome analyses have been conducted on switchgrass (Panicum virgatum L.), which is another important bioenergy crop widely used for lignocellulosic researches. Comparative transcriptome analyses of different switchgrass ecotypes identi ed many molecular biomarkers of phenotypic traits, which accelerated the development of highyielding switchgrass cultivars [10,11]. Although several Miscanthus species have been considered important feedstocks for biofuels and biore neries, their genetic resources, including transcriptome data, are currently very limited.…”