TFs play important roles in plants and are involved in diverse stress signaling pathways through activation or inhibition of target gene expression. Recent research has explored the various functions of GATA TFs in rice, but their impact on cold tolerance has not been explored. Informatic and expression analysis of rice OsGATA family proteins demonstrated their involvement with abiotic stress responses, and several of the genes showed duplicated relationships and similar expression patterns during rice growth (Gupta et al. 2017). Similarly, GATA proteins in the Chickpea were shown to be involved in the response to ABA and Drought stress (Niu et al. 2020). These recent studies into GATA TFs suggest that members of the GATA gene family may be generally involved in responses to abiotic stress. In this study, a novel transcription factor, OsGATA16, was identified that increased cold tolerance in rice with no apparent impact on agronomic growth traits under field conditions. These results are consistent with previous assessments of GATA-family members and support the hypothesis of a broader role for this gene family in abiotic stress responses.
OsGATA16 was ubiquitously expressed in rice tissues, with the highest expression levels in panicles (Fig. 3a), indicating that OsGATA16 might be involved in the cold response as well as in the response to a range of abiotic stresses and phytohormones through association with other factors. Transcriptional analysis by qRT-PCR showed that OsGATA16 expression was induced by cold and ABA treatments, and was suppressed by drought, BA, and JA treatments (Fig. 2). Several cis-acting elements were found in the OsGATA16 promoter (Table 1), as well as a range of TF binding sites such as WRKY, MYB, ABRE, and bHLH. A transcriptional study of OsMyb4, a Myb TF involved in responses to stress, highlighted a regulatory network that facilitated the cold stress signaling pathway through mediator MYB, bZIP, NAC, ARF, ERF, and CCAAT-HAP TFs. Osmyb4 overexpression also impacted panicle development, and OsGATA16 expression increased 3.1-fold in overexpression lines (Park et al. 2010). OsRAN1, an evolutionarily conserved member of the small G-protein family, was found to have a significant role in improving cold tolerance in rice. Like OsGATA16, OsRAN1 was also expressed ubiquitously in rice tissue and exhibited highest expression in panicles (Xu and Cai 2014). These studies are consistent with the findings that OsGATA16 overexpression conferred improved cold tolerance, and that the highest OsGATA16 expression was found in panicles. Together, this suggests that OsGATA16 may associate with other TFs in panicle tissues to mediate responses to cold exposure as well as to other abiotic stresses and phytohormones.
OsGATA16 localized to the cell nucleus and acted as a transcription repressor. Transcriptional analysis of cold-sensitive genes in OsGATA16-overexpressing (OE) and WT lines by qRT-PCR revealed that OsWRKY45-1, OsSRFP1, OsCYL4, and OsMYB30 transcription was repressed in OE lines compared with WT. Yeast one-hybrid and Dual-luciferase reporter assays confirmed that OsGATA16 bound to and suppressed the activity of the OsWRKY45-1 promoter. Previous research reported the involvement of OsWRKY45-1 in the response to low temperatures (Tao et al. 2011). OsWRKY45-1 and OsWRKY45-2 (alleles of OsWRKY45) played different roles in the response to ABA and salt stress, but showed similar sensitivities to cold and drought stress (Tao et al. 2011). This suggests that OsGATA16 improves cold tolerance in rice by repressing the expression of the cold-sensitive gene OsWRKY45-1. Recent research identified several novel functions for OsWRKY45, and it is thus possible that OsGATA16 repression of OsWRKY45-1 expression is involved in other biological functions in addition to the cold response. The OsWRKY45-1 and OsWRKY45-2 alleles encode proteins that differ by ten amino acids, and several reports associate OsWRKY45 (OsWRKY45-1 or OsWRKY45-2) with disease in rice (Tao et al. 2009). The two alleles exhibited contrasting roles in resistance to bacterial blight caused by Xoo and bacterial leaf streak caused by Xoc. Overexpression of OsWRKY45-1 reduced resistance to Xoo and Xoc but increased resistance to rice blast disease, caused by the fungus Magnaporthe grisea. The response to Xoo infection was accompanied by increased accumulation of SA and JA (Tao et al. 2009). In this study, expression of OsGATA16 was repressed by JA exposure, suggesting that OsGATA16 may act as a positive regulator of disease resistance: upon infection, elevated JA levels would suppress OsGATA16 expression and lead to de-repression of OsWRKY45-1, increasing resistance to M. grisea but decreasing resistance to Xoo and Xoc. Another regulatory factor, OsNPR1, also affected disease resistance in rice. Overexpression of OsNPR1 conferred disease resistance to bacterial blight (Yuan et al. 2007), and OsNPR1 was found to repress the accumulation of OsbHLH6 in the cell nucleus, thereby repressing JA signaling (Meng et al. 2020). Microarray analysis of rice transcription during Xoo infection showed that OsGATA16 expression decreased upon infection (Kong et al. 2020). Taken together, these results suggest that OsGATA16 might act as a regulator of Xoo, Xoc, and M. grisea resistance. We propose that the bacterial blight resistance associated with OsNPR1 overexpression (Yuan et al. 2007) and OsbHLH6 consumption in the cell nucleus (Meng et al. 2020) occurred as a result of increased expression of OsGATA16 due to repression of JA signaling. The decrease in OsGATA16 expression after Xoo expression (Kong et al. 2020) further suggests that OsGATA16 plays an antagonistic role against Xoo. Furthermore, OsGATA16 was induced by ABA treatment (Fig. 2c), and ABA signaling was negatively regulated by OsWRKY45-1 and positively regulated by OsWRKY45-2 (Tao et al. 2011), suggesting that repression of the OsWRKY45-1 promoter by OsGATA16 may involve the ABA signaling pathway. Further analysis is needed to confirm the mechanisms by which OsGATA16 mediates disease resilience.
GATA-family TF proteins are highly conserved. Most family members retain a GATA-type zinc finger protein domain proximal to the DNA-binding domain, with a zinc finger protein domain also involved in identifying GATA TF recognition sequences (Behringer and Schwechheimer 2015). Several functions of GATA-family TFs have been identified, including functions associated with cytokinin, nitrate, and light responses, and with chloroplast development and plant growth. Responses of the family are diverse, as illustrated by Cga1 (OsGATA11), which was induced by cytokinin (Hudson et al. 2013), in contrast to the repression of OsGATA16 by cytokinin. This may indicate antagonistic functions for GATA-family members in cytokinin mechanisms, or may suggest the involvement of different GATA-family members in the transcriptional regulation of different signaling pathways.
Rice subspecies Japonica and Indica exhibit polarization for many agronomic traits, including adaptation and resilience to low temperatures (Ma et al. 2015). Japonica varieties generally display better tolerance to cold stress than Indica varieties, due to evolutionary adaptations to growth in regions with different climates (Wang et al. 2014). Some cold-related genes may have retained their ancestral functions in older varieties, but environmental adaptations may have supported the persistence of novel alleles with different functions in cultivated rice varieties that have been further selected and preserved by breeding processes (Kim et al. 2016). For example, OsbZIP73, which is involved in the ABA-dependent cold signaling pathway, harbors a single SNP between Japonica and Indica varieties. The SNP is located in an exon and leads to an amino acid disparity that partially explains differences in cold tolerance between subspecies (Liu et al. 2018). In another study, an SNP (SNP2) in COLD1, a novel cold sensor in rice, was highly variable among diverse subspecies, but was conserved in Japonica varieties and was associated with cold tolerance in cultivated rice (Ma et al. 2015). In this study, haplotype analysis of the OsGATA16 gene detected novel alleles associated with different subspecies. Eleven SNPs were identified within a strong LD block, five Haps were distinguished according to SNP variation, and Japonica and Indica varieties were clearly defined in two separate groups. Phenotypic analysis showed that the Indica group was significantly more cold-sensitive than the Japonica group. A non-synonymous functional SNP (SNP 8, 336A/G) was significantly associated with cold tolerance in both Japonica and Indica varieties when considered separately or together. As with OsbZIP73 and COLD1, OsGATA16 showed clear differentiation between rice subspecies and conferred cold tolerance in rice. Furthermore, the 336G allele was significantly associated with cold tolerance in both Indica and Japonica varieties, and has potential as a novel functional allele for improving cold resilience in rice breeding programs.