Influence of isopropylmalate synthase OsIPMS1 on seed vigour associated with amino acid and energy metabolism in rice

Abstract

Seed vigour is an imperative trait for the direct seeding of rice. Isopropylmalate synthase (IPMS) catalyses the committed step of leucine (Leu) biosynthesis, but its effect on seed vigour remains unclear. In this study, rice OsIPMS1 and OsIPMS2 was cloned, and the roles of OsIPMS1 in seed vigour were mainly investigated. OsIPMS1 and OsIPMS2 catalyse Leu biosynthesis, and Leu feedback inhibits their IPMS activities. Disruption of OsIPMS1 resulted in low seed vigour under various conditions, which might be tightly associated with the reduction of amino acids in germinating seeds. Eleven amino acids that associated with stress tolerance, GA biosynthesis and tricarboxylic acid (TCA) cycle were significantly reduced in osipms1 mutants compared with those in wide type (WT) during seed germination. Transcriptome analysis indicated that a total of 1209 differentially expressed genes (DEGs) were altered in osipms1a mutant compared with WT at the early germination stage, wherein most of the genes were involved in glycolysis/gluconeogenesis, protein processing, pyruvate, carbon, fructose and mannose metabolism. Further analysis confirmed that the regulation of OsIPMS1 in seed vigour involved in starch hydrolysis, glycolytic activity and energy levels in germinating seeds. The effects of seed priming were tightly associated with the mRNA levels of OsIPMS1 in priming seeds. The OsIPMS1 might be used as a biomarker to determine the best stop time-point of seed priming in rice. This study provides novel insights into the function of OsIPMS1 on seed vigour and should have practical applications in seed priming of rice.

Keywords: Oryza sativa; isopropylmalate synthase; seed priming; seed vigour.

Figure 1

Activities of OsIPMS1 and OsIPMS2 in rice. (a) Pathway for Leu biosynthesis. (b) Purification of the expressed GST‐Tag OsIPMS1 and OsIPMS2 protein. (c, d) OsIPMS1 and OsIPMS2 activities were identified by directly measuring absorbance at 412 nm. (e,f) The activities of OsIPMS1 and OsIPMS2 are subjected to Leu feedback inhibition. ** indicates the significant difference at 1% level.

Figure 2

Expression patterns of OsIPMS1 and OsIPMS2 in rice. Expression pattern of OsIPMS1 and OsIPMS2 in various developmental stages (a) and tissues (b) of rice based on the publicly available microarray data ( http://www.genevestigator.com). Transcription levels of OsIPMS1 and OsIPMS2 in filling grains (c) and germinating seeds (d) were conducted using qRT‐PCR approach. The expression of OsIPMS1 and OsIPMS2 was normalized to that of OsActin gene control. The relative expression levels were represented by fold change relative to the expression level of OsIPMS1 at 0 DAF (c) or 4‐h imbibition stage (d). Each column represents the means ± standard deviation.

Figure 3

Comparison of seed germination between WT and osipms1 mutants under normal conditions. (a) Gene structure of OsIPMS1 with a T‐DNA insertion and locations of the primers used for PCR analysis. Triangle represents the T‐DNA. Green and white boxes represent exons and UTR of OsIPMS1, respectively. Solid lines represent introns. F, R and T‐DNA‐F primers used for genotyping PCR. (b) PCR of genomic DNA from WT and osipms1a. qRT‐PCR analysis of OsIPMS1 in WT and osipms1a seeds. The expression of OsIPMS1 was normalized to that of OsActin gene control. The relative expression levels were represented by fold change relative to the expression level of WT. (c) One nucleotide was added in the osipms1b and osipms1c mutants and produced a premature stop. (d) Seed germination of WT and osipms1 mutants after 3 and 5 days. Bars = 10 mm. (e) germination potential; (f) time to 50% germination percentage; (g) germination index; (h) length of shoots and roots; (i) fresh weight of shoots and roots; (j) dry weight of shoots and roots. Each column represents the means ± standard deviation. * and ** indicate the significant difference compared to WT at 5% and 1% levels, respectively. n.s. represents not significant.

Figure 4

Comparison of amino acid levels between WT and osipms1 mutants during seed germination. Each point represents the means ± standard deviation. * and ** indicate the significant difference compared to WT at 5% and 1% levels, respectively.

Figure 5

Comparison of seedling growth between WT and osipms1 mutants under amino acids treatments. (a) Seedling growth in WT and osipms1 mutants under normal and amino acids treatment conditions for 5 days. Bars = 10 mm. Comparison of shoot and root length between normal and amino acids treatment in WT (b) and osipms1 mutants (c, d, e). Each column represents the means ± standard deviation. ** indicates the significant difference compared to normal condition at 1% level. n.s. represents not significant.

Figure 6

OsIPMS1 altering expression of genes involved in glycolysis. (a) Differentially expressed genes (DEGs) involved in glycolysis. (b) Comparison of OsPKs, OsPFKs and OsPEPCK expression between WT and osipms1 at 8‐h imbibition stage using qRT‐PCR approach. The expression of genes was normalized to that of OsActin gene control. The relative expression levels were represented by fold change relative to the expression level of WT. Each metabolism column represents the means ± standard deviation. ** indicates the significant difference compared to WT at 1% level. n.s. represents not significant.

Figure 7

OsIPMS1 altering seedling growth involved in GA biosynthesis. (a) GA ₃ content in WT and osipms1 mutants during seed germination. (b) General overview of GA biosynthesis pathway in rice according to previous reports. (c) Relative expression levels of GA biosynthesis‐related genes in germinating seeds. The expression of genes was normalized to that of OsActin gene control. The relative expression levels were represented by fold change relative to the expression level of OsKAO in WT. (d) Seedling growth in WT and osipms1 mutants under normal and GA ₃ treatment conditions for 5 days. Bars = 10 mm. Comparison of shoot length (e) and root length (f) between WT and osipms1 mutants under normal and GA ₃ treatment conditions. Each column represents the means ± standard deviation. * and ** indicate the significant difference compared to WT at 5% and 1% levels, respectively. n.s. represents not significant.

Figure 8

Comparison of starch mobilization between WT and osipms1 mutants during seed germination. (a) α‐amylase activity; (b) β‐amylase activity; (c) glucose content; (d) fructose content. Each column represents the means ± standard deviation. * and ** indicate the significant difference compared to WT at 5% and 1% levels, respectively. n.s. represents not significant.

Figure 9

Comparison of glycolytic activity and energy levels between WT and osipms1 mutants during seed germination. (a) pyruvate acid content; (b) acetyl‐CoA content; (c) ATP content; (d) ADP content; (e) AMP content; (f) energy charge. Each column represents the means ± standard deviation. * and ** indicate the significant difference compared to WT at 5% and 1% levels, respectively. n.s. represents not significant.

Figure 10

Relationship between priming effects and OsIPMS1 expression in primed seeds of rice Ningdao NO.1 and Wuyungeng NO.7. (a) Comparison of seedling growth among various durations of priming treatments. Bars = 10 mm. (b) germination potential; (c) seedling percentage. (d,e) Transcription levels of OsIPMS1 during the priming process. The expression of OsIPMS1 was normalized to that of OsActin gene control. The relative expression levels were represented by fold change relative to the expression level of OsIPMS1 at 4‐h priming stage. Each column represents the means ± standard deviation. Different lowercase letters represent significant difference at 5% level.

Figure 11

Hypothetical model of the role of OsIPMS1 on seed vigour in rice. The expression of OsIPMS1 in germinating seeds induces the accumulation of the amino acids associated with stress tolerance, GA biosynthesis and TCA cycle. Increased GA biosynthesis enhances starch hydrolysis for the accumulation of soluble sugars, thus increasing the glycolytic activity. The ATP levels will be promoted through TCA cycle with the increases of glycolysis and TCA cycle metabolites, which contribute to rapid germination and vigorous seedling growth. Solid arrows indicate the major effects, while dashed arrows indicate the predicted minor effects.