Mutation of the OsGlyRS3 gene affects heading date in rice

ABSTRACT

Aminoacyl-tRNA synthetases play a critical role in protein synthesis by catalyzing the covalent attachment of amino acids to their cognate tRNAs. However, the role of aminoacyl-tRNA synthetases in the transition from vegetative to reproductive growth in plants remains poorly understood. In this study, a rice (Oryza sativa) glycyl-tRNA synthetase 3, OsGlyRS3, was found to impact heading date in rice. Flowering in osglyrs3, a mutant line containing a T-DNA insertion in OsGlyRS3, was advanced by approximately 2 weeks compared to wild type. Expression analysis of flowering regulator genes showed that transcript levels of Heading date 1 (Hd1), Heading date 3a (Hd3a), and OsMADS51 were elevated in osglyrs3. These data indicate that the loss of OsGlyRS3 activity induces the expression of flowering-activating genes, resulting in early flowering.

KEYWORDS:

• OsGlyRS3
• rice
• flowering
• glycyl-tRNA synthetase
• Hd3a

Result and discussion

The timing of floral transition is a key determinant of rice adaptation and production. Because flowering time also affects other agronomic traits, the molecular mechanisms underlying the timing of the transition from vegetative to reproductive growth have been extensively studied in rice.^1,2,3 As a result, several flowering-related genes, including OsGIGANTEA (OsGI, a rice homolog of Arabidopsis GIGANTEA gene), Hd1 (a rice ortholog of Arabidopsis CO gene), Hd3a (a rice ortholog of Arabidopsis FT gene), OsMADS50 (a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)), OsMADS51 (a type I MADS-box gene), GRAIN NUMBER PLANT HEIGHT AND HEADING DATE 7 (Ghd7, a CCD domain transcription factor), and EARLY HEADING DATE 1 (Ehd1, a B-type response regulator), have been identified in rice.^{1,4,5,6,7,8,9,10,11} These flowering-related genes regulate heading date under short-day (SD) and/or long-day (LD) conditions.

GlyRS catalyzes the covalent attachment of glycine to its cognate tRNA. Arabidopsis EMBRYO DEFECTIVE DEVELOPMENT1 (EDD1) is a nuclear gene that encodes a glycyl-tRNA synthetase that localizes to plastids and mitochondria, and loss-of-function mutations in EDD1 are embryonic lethal.^12,13,14 In addition, edd1 mutants also display abnormal leaf development and patterning.¹⁵ In rice, the Rice Albino 1 (RA1) gene (LOC_Os06g01400) encodes a GlyRS that localizes to chloroplasts. The ra1 mutant has decreased chlorophyll and carotenoid levels as well as plastidic structural defects, including abnormal thylakoid membrane structures and increased numbers of osmiophilic particles, which produce albino phenotypes in seedlings.¹⁶

Previously, we examined proteins that accumulated specifically in the seeds of a low eating-quality rice cultivar, Dobong, and a high eating-quality cultivar, Gopum, and found that OsGlyRS3 accumulated in immature seeds of Dobong and accumulated at high levels in mature seeds of both Dobong and Gopum.¹⁷ However, OsGlyRS3 levels in mature seeds were two-fold higher in Gopum than in Dobong seeds, suggesting that OsGlyRS3 might be a positive regulator of high eating quality. Thus, we further examined the effect of OsGlyRS3 on vegetative and reproductive growth during rice development.

The effects of OsGlyRS3 were investigated using mutant analysis of the encoding gene, OsGlyRS3 (LOC_ Os08g42560). The OsGlyRS3 gene contains nine exons and eight introns (Figure 1(a), upper), and the OsGlyRS3 protein contains three conserved domains: a plant-specific helix-turn-helix WHEP domain (57–115 aa), a glycyl-tRNA synthetase-like core domain (130–476 aa), and an anticon binding domain (627–715 aa) (Figure 1(a), lower). An OsGlyRS3 mutant was identified from T-DNA insertion mutant lines (provided by the GynHeung An laboratory, Kyung Hee University, Korea). The mutant (PFG-3A-53125) harbored a T-DNA insertion in the fifth intron of the OsGlyRS3 gene, and was named osglyrs3 (Figure 1(a), upper). Growth was assessed in Dongjin (wild type, WT) and osglyrs3 (T-DNA insertion mutant) plants. No significant differences were observed between WT and osglyrs3 during the germination and vegetative phases, although osglyrs3 appeared to grow slightly faster than WT. However, substantial differences were observed during the heading stage. Flowering time was estimated using days-to-heading, as determined by the appearance of the first panicle. Flowering in osglyrs3 mutants occurred approximately 2 weeks earlier than in WT plants (Figure 1(b and c)), indicating that loss of OsGlyRS3 induced the transition from the vegetative to the reproductive phase. Early flowering in osglyrs3 also suggested that the expression of flowering-related genes was upregulated or downregulated in the mutant. The expression of five flowering-related genes, OsGI, Hd1, Hd3a, OsMADS50, and OsMADS51 genes was investigated using RT-PCR with gene-specific primers. Transcript levels of OsGI and OsMADS50 were similar between WT and osglyrs3, but expression of Hd1, Hd3a, and OsMDS51 was upregulated in osglyrs3.

Figure 1. Early flowering in the osglyrs3 rice mutant. (a) Schematic representation of OsGlyRS3. The OsGlyRS3 (Os08g42560) gene consists of nine exons (boxes) and eight introns (lines between boxes). The osglyrs3 mutant (PFG-3A-53125) contains a T-DNA insertion in the fifth intron (upper). The OsGlyRS3 protein (lower) contains a helix-turn-helix WHEP domain, a glycyl-tRNA synthetase-like core domain, and an anticon binding domain. (b) Phenotypes of wild-type (WT) and osglyrs3 mutant plants at the heading stage under long day (LD; 13.5 light/10.5 dark) conditions in the field. (c) Heading date in osglyrs3 and WT plants under LD conditions. Error bars indicate standard deviations. DAG, days after germination. Asterisks denote P-value <0.01. (d) Expression of floral regulators in osglyrs3 mutant plants. Total RNAs were prepared from second leaf blades of WT and osglyrs3 plants grown under LD conditions. Transcript levels of the OsGI, Hd1, Hd3a, OsMADS50, and OsMADS51 genes were assessed using RT-PCR with gene-specific primers. OsGI, a rice ortholog of the Arabidopsis GIGANTEA gene, Hd1, a rice ortholog of the Arabidopsis CO gene; Hd3a, a rice ortholog of the Arabidopsis FT gene; OsMADS50, a rice ortholog of the Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene; and OsMADS51, a type I MADS-box gene involved in the SD promotion pathway in rice

Previous research showed that OsGI upregulated the expression of Hd1, which activated expression of Hd3a, promoting rice heading under both short day (SD) and long day (LD) conditions.^18,19 OsMADS50 acted as an upstream regulator of Hd3a and stimulated flowering,⁶ and OsMADS51 was also shown to act as a flowering promoter, particularly under SD conditions.⁷ Mutation of the OsGI gene was found to significantly delay flowering under SD conditions and slightly delay flowering under LD conditions.¹¹ However, other studies showed that hd1-1 mutants experienced late flowering under SD conditions and early flowering under LD conditions,^9,20 prompting the hypothesis that Hd1 acted as a flowering activator under SD conditions and as a flowering repressor under LD conditions. Notably, Hd1 overexpression caused constitutive late flowering under both SD and LD conditions,²¹ suggesting that Hd1 bound to different partners under different conditions.

In this study, OsGI and OsMADS50 expression was not affected by the loss of OsGlyRS3 activity, whereas Hd1, Hd3a, and OsMADS51 transcript levels were substantially elevated in osglyrs3, suggesting an additional mechanism or pathway for the regulation of OsGlyRS3-mediated gene expression. Taken together, our findings and results from earlier research suggest the existence of other regulatory pathways involved in modulating flowering time in rice.

Two genes encoding GlyRS were identified in the rice genome (http://ricexpro.dna.affrc.go.jp): OsGlyRS1 (LOC_Os04g32650) and OsGlyRS3. OsGlyRS1 was highly expressed in leaf and inflorescence tissues, and OsGlyRS3 was mainly expressed in ovaries and anthers, but no information regarding subcellular localization was available. Recent research indicated that RA1 was also a GlyRS and was involved in chloroplast development,¹⁶ but RA1 was not homologous to OsGlyRS1 and OsGlyRS3 at the amino acid level. Further studies, such as expression analysis of OsGlyRS1 and OsGlyRS3 and assessment of the effects of OsGlyRS1 and OsGlyRS3 on tissue and organ development, will help elucidate their roles in vegetative and reproductive growth.

Disclosure of potential conflicts of interest

The authors report no conflict of interest.

Additional information

Funding

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01567701)” Rural Development Administration, Republic of Korea.