Elucidation of the miR164c-Guided Gene/Protein Interaction Network Controlling Seed Vigor in Rice

Abstract

MicroRNAs (miRNAs) play important roles in various aspects of plant physiology and metabolism. The expression level of miR164c is negatively correlated with seed vigor in rice ( Oryza sativa L.); however, the mechanism of seed vigor regulation by miR164c remains unknown. Anti-aging capacity is an important indicator of seed vigor. Here, we report an miR164c-guided gene/protein interaction network that regulates the anti-aging ability of rice seeds. Seeds of the wild-type (WT) rice cultivar “Kasalath” and its transgenic derivatives, miR164c-silenced line (MIM164c) and miR164c overexpression line (OE164c), with significant differences in anti-aging capacity, showed significant differences in gene and protein expression levels. The differentially expressed genes (DEGs) or proteins were significantly enriched in six metabolic functional categories related to seed vigor, including “stress response,” “protein processing in endoplasmic reticulum (ER),” “embryo development,” “serine-type endopeptidase inhibitor,” “energy metabolism,” and “other.” Differences in the expression levels of genes or proteins related to energy metabolism, serine endopeptidase, and stress response in seeds under normal storage conditions may be associated with anti-aging capacity. The results of gene/protein interaction analyses suggest that miR164c first targets PSK5 , and the PSK5 protein then interacts with the ubiquitin-associated gene RPS27AA , which simultaneously impacts the genes/proteins in the six above-mentioned functional categories. Expression levels of some of the key genes and proteins in the interaction network were verified by real-time fluorescence quantitative PCR (RT-qPCR) and multiple reaction monitoring mass spectrometry (MRM-MS), respectively. Thus, the present study provides new insights into the miRNA-mediated gene and protein interaction network that regulates seed vigor.

Degradome sequencing reveals an integrative miRNA-mediated gene interaction network regulating rice seed vigor

“…Seed germination rate is an important indicator of seed vigor. The results of RT-qPCR analysis and germination test indicated that after 8 days of artificial aging, the expression level of miR164c and germination rates of WT, MIM164c and OE164c seeds differed significantly, consistent with previous studies [ 10 , 11 ]. Regardless of aging, OE164c seeds showed the highest miR164c expression level and the lowest germination rate, whereas MIM164c seeds displayed the lowest miR164c expression level and the highest germination rate (Fig.…”

“…In addition to the previously reported miR164c-guided RPS27AA -related pathway [ 11 ], the network contained at least other seven KEGG pathways: ‘ascorbate and aldarate metabolism’ ‘plant hormone signal transduction’ ‘galactose metabolism’ ‘nucleotide excision repair’ ‘TCA cycle’ ‘oxidiative phosphorylation’ and ‘flavonoid biosynthesis’ (Fig. 4 ), of which the RPS27AA -related pathway and three KEGG pathways that directly interact with the target genes of miR164s were simplified as Fig.…”

“…To further explore the molecular mechanism of seed vigor regulation by miRNAs, we analyzed the interactions among all target genes of miRNAs identified in the present study using the STRING database, an online database resource search tool for retrieving interacting genes, comprehensively covering relevant experimental and predicted gene interaction information. We also analyzed genes corresponding to the six metabolic functional categories of rice seed vigor related differentially expressed proteins (DEPs) reported in Huang et al (2020) [ 11 ] as well as genes corresponding to DEPs among artificially aged WT, MIM164c , and OE164c seeds (1.3 < FC < 1/1.3). The results revealed an interaction network (Fig.…”

“…Inhibition of miR164c expression can improve the vigor and anti-aging ability of rice seeds [ 10 ]. Additionally, miR164c affects the key gene RPS27AA by acting on target genes OsPSK5 and TIL1 ( OMTN2 ), which then affects energy metabolism-, endoplasmic reticulum (ER)-, stress-, and embryo development-related proteins, serine endopeptidase inhibitors and others, ultimately regulating rice seed vigor [ 11 ]. However, whether and how other miRNAs cooperate with miR164c to regulate seed vigor remains unknown.…”

Quantitative Transcriptomic and Proteomic Analysis of Fruit Development and Ripening in Watermelon (Citrullus lanatus)

“…Finally, the peptides were combined into 6 fractions and dried by a vacuum-type centrifuge. LC-MS/MS Analysis and Database Search were conducted according to the previously published method ( Huang et al, 2020 ). Briefly, we subjected the peptides to an NSI source, followed by MS/MS in Q ExactiveTM (Thermo Fisher Scientific, Waltham, MA, United States) coupled with UPLC online.…”