Identification and Comparative Analysis of Cadmium Tolerance-Associated miRNAs and Their Targets in Two Soybean Genotypes

Abstract

MicroRNAs (miRNAs) play crucial roles in regulating the expression of various stress responses genes in plants. To investigate soybean (Glycine max) miRNAs involved in the response to cadmium (Cd), microarrays containing 953 unique miRNA probes were employed to identify differences in the expression patterns of the miRNAs between different genotypes, Huaxia3 (HX3, Cd-tolerant) and Zhonghuang24 (ZH24, Cd-sensitive). Twenty six Cd-responsive miRNAs were identified in total. Among them, nine were detected in both cultivars, while five were expressed only in HX3 and 12 were only in ZH24. The expression of 16 miRNAs was tested by qRT-PCR and most of the identified miRNAs were found to have similar expression patterns with microarray. Three hundred and seventy six target genes were identified for 204 miRNAs from a mixture degradome library, which was constructed from the root of HX3 and ZH24 with or without Cd treatment. Fifty five genes were identified to be cleaved by 14 Cd-responsive miRNAs. Gene ontology (GO) annotations showed that these target transcripts are implicated in a broad range of biological processes. In addition, the expression patterns of ten target genes were validated by qRT-PCR. The characterization of the miRNAs and the associated target genes in response to Cd exposure provides a framework for understanding the molecular mechanism of heavy metal tolerance in plants.

Genetic Control of Cadmium Concentration in Soybean Seeds

“…Of these 26 miRNAs identified, gma-miR1535b, which was detected as being up-regulated in HX3 and down-regulated in ZH24 and all other miRNA, showed similar expression patterns in HX3 and ZH24. This suggested that miRNA regulation may represent the fundamental mechanism of adapting to Cd exposure [94]. Further, it was reported that miR397a, miR408, and miRNA398c showed almost the similar up-regulated alteration in response to Cd exposure, which might imply that SPL7 (SQUAMOSA promoter-binding protein-like 7) is involved in the regulation of Cu deficiency and Cd response in soybean [94].…”

“…CK was reported to inhibit primary root elongation in A. Thaliana [103]. Under Cd exposure, Glyma07g38620.1 displayed an apparent up-regulation in HX3 and slight down-regulation in ZH24, so does the CK, which probably explain why HX3 show higher tolerance and distinctly primary root elongation inhibition than that of ZH24 [94].…”

“…Under Cd stress, 26 Cd-responsive miRNAs were found [94]. Of these 26 miRNAs identified, gma-miR1535b, which was detected as being up-regulated in HX3 and down-regulated in ZH24 and all other miRNA, showed similar expression patterns in HX3 and ZH24.…”

“…This suggested that miRNA regulation may represent the fundamental mechanism of adapting to Cd exposure [94]. Further, it was reported that miR397a, miR408, and miRNA398c showed almost the similar up-regulated alteration in response to Cd exposure, which might imply that SPL7 (SQUAMOSA promoter-binding protein-like 7) is involved in the regulation of Cu deficiency and Cd response in soybean [94]. To evaluate the target transcripts of the miRNAs, a high-throughput degradome sequencing was adopted using a small RNA library.…”

“…Fifty-five targets cleaved by 14 Cd-responsive miRNAs were identified. In addition, a number of Cd-responsive miRNAs and target mRNAs in soybean have been validated by quantitative RT-PCR [94]. It is well established that lignin provides structural support and contributes to plant defense mechanism against both biotic and abiotic stresses [95].…”