Elucidation of the molecular mechanisms for overwintering phenotype of Lotus japonicus controlled by natural variation.

概要

Introduction
As sessile organisms, plants must cope with their environmental conditions and thus have evolved a set of perception and signaling mechanisms to respond or adapt to adverse environmental conditions. Colonization of the new habitats is expected to require modification of these mechanisms to overcome challenges in new environmental factors. Temperature is one of the environmental factors that affect plant growth and development. Thus, adaptation to the low temperature is essential for long term evolutionary success of plants, especially for those in temperate region (Körner, 2016). For perennial plants, the cold tolerance, in other words, winter- hardiness is fundamental mechanism for surviving during winter season.

In general, cold tolerance in many plant species is controlled by multiple genes and complex biological processes (Preston and Sandve, 2013). The process of gaining cold tolerance by low temperature stress that involved in both biochemical and physiological changes is known as cold acclimation. In the state of cold acclimation, stabilization of cell wall and membrane are induced by modification of metabolic processes, such as carbohydrate and lipid, through changing the regulation of sets of genes (Wingler, 2005; Guo et al., 2018; Liu et al., 2019). Among the gene expression regulation pathways involved in cold acclimation, ICE-CBF-COR pathway is the most characterized pathways mainly analyzed in Arabidopsis. Although the components of ICE-CBF-COR pathway are conserved in perennial plants, the response to long term cold treatment has shown to be different between perennial plants and Arabidopsis. Thus, the detailed analysis of responses against low temperature in perennial plant, especially overwintering phenotype as its characteristic trait, will provide the new insights of cold stress responses in plants. Lotus japonicus is a perennial legume plant, which have been used as experimental model of legume plants with a variety of material resources, including a collection of Japanese wild accessions facilitated to carry out GWAS analysis. Based on the accumulated SNPs information, it was speculated that wild accessions of L. japonicus were colonized in Japan around 25,000 years ago in Kyushu island, then divided into three subpopulation and gradually spread dispersed to high latitude area along the end of glacial era (~10,000 years ago) (Shah et al., 2020). Considering this estimation, wild accessions of L. japonicus is considered to be a suitable for the analysis on local adaptation based on overwintering trait, and field experiment in Tohoku University field would also fit for the evaluation of overwintering phenotype. With the aim to elucidate the molecular mechanism of overwintering trait in perennial plant with a relation to local adaptation, I carried out molecular genetic approaches by using 120 wild accessions of L. japonicus.

Results and Discussions
- Phenotypic variation and GWAS for overwintering phenotype of L. japonicus in Tohoku field.
A total of 120 wild accessions of L. japonicus were grown in Tohoku University field in Kashimadai, Miyagi prefecture. I found significant differences of overwintering phenotypes among the wild accessions with strong latitude dependency as well as subpopulation dependency. Furthermore, the northern accessions displayed higher winter survival rates than southern accessions, and accessions classified in subpopulation 3 displayed higher winter survival rates than the other subpopulations. As a result of GWAS using overwintering phenotype in 2014-2015, two genomic loci were displayed a highly significant association with the phenotypes, and members of receptor-like protein kinases (RLKs), LjFER and LjSRK, were identified in these genetic loci on chr1 and chr6, respectively. LjFER encodes a protein very similar to the Arabidopsis FERONIA receptor-like kinase, and LjSRK encodes a putative G-type lectin S-receptor like serine/threonine protein kinase. On LjFER, six SNPs are identified in the 5’UTR, which compose two haplotypes LjFER_Ref and LjFER_Alt in Japanese wild accessions of L. japonicus analysed so far. Both of the haplotypes were identified in three subpopulations, with LjFER_Ref is a majority in subpopulation 1 and subpopulation 2, while LjFER_Alt associate with higher survival rate (winter- hardiness) in the majority of subpopulation 3. On LjSRK, three SNPs are identified on coding region, two of which are non-synonymous. Three haplotypes, LjSRK_Hap1, LjSRK_Hap2 and LjSRK_Hap3, are composed of these SNPs, of which LjSRK_Hap2 and LjSRK_Hap3 associated winter-hardiness are identified only in the subpopulation 3, suggesting that mutations causing LjSRK_Hap2 and LjSRK_Hap3 occurred after subpopulations were established. Additionally, these detected genomic loci were identified as genome regions with high fixation index (FST) level in comparison of subpopulation 3 occupied northern Japan and other subpopulations. This overlap between top GWA SNPs for overwintering and genome-wide top FST signal indicates that winter-hardiness haplotypes in LjFER and LjSRK provided adaptive roles during colonization to higher latitude in Japan that has low temperature in winter.

- Functional analysis of LjFER and LjSRK in winter-hardiness using LORE1 insertion tag lines
To investigate the roles of LjFER and LjSRK in winter-hardiness, the functional analysis was carried out using L. japonicus retrotransposon (LORE1) insertion tag lines. As the result, the phenotypes evaluation of homozygous LjSRK_LORE1 confirmed the gene function in winter-hardiness. For LjFER, I could not obtain the homozygous insertion lines, however, I could test the phenotype since significant reduction of gene expression level was detected among the heterozygous LjFER_LORE1 lines. Thus, it was confirmed that each of LjFER and LjSRK contributed in establishing of winter-hardiness in wild accessions of L. japonicus.

- Effect of haplotypes of LjFER and LjSRK in winter-hardiness in Tohoku field and greenhouse experiment
To make detailed analysis of haplotype effects of LjFER and LjSRK to the winter-survival in Tohoku field, statistical comparisons were performed using the phenotypes in the Tohoku field observed from 2014 to 2020. By this comparative analysis of the accessions with different combination of haplotypes of LjFER and LjSRK, the effect of each winter-hardiness haplotype was confirmed in Tohoku field and additive effect of two winter-hardiness haplotypes was also confirmed. In comparison between the accessions with LjFER_Ref/LjSRK_Hap1 and LjFER_Alt/LjSRK_Hap1, higher survival rate was observed in accessions with winter- hardiness haplotype, LjFER_Alt in LjFER with significant differences. Similarly, in comparison between the accessions with LjFER_Alt/LjSRK_Hap1 and LjFER_Alt/LjSRK_Hap3, higher survival rate was observed in accessions with additional winter-hardiness haplotype, LjSRK_Hap3 with statistical significance. These results indicated that each of winter-hardiness haplotypes LjFER_Alt and LjSRK_Hap3 has potential to increase the winter-survival rate in Tohoku field, and there is an additive effect of these two winter-hardiness haplotypes. Further analysis of haplotype effect of LjFER and LjSRK in winter-hardiness was carried out in greenhouse by using 23 wild accessions selected by considering the haplotypes combination as well as geographical and subpopulation relationship. The greenhouse experiment repeated weekly from late December to the early April, in which 5 weeks- old plants grown in greenhouse were placed in natural winter environment for 4 weeks then returned to greenhouse for two weeks to evaluate the phenotype. These result indicated that winter-hardiness haplotypes of LjFER and LjSRK increase the survival rate especially in middle of experimental period (mid-winter). Moreover, the greenhouse experiment also revealed the effect of low temperature level, latitude distributions, and L. japonicus subpopulations to survival determination of L. japonicus.

- The haplotype dependency in expression level of LjFER and LjSRK and their correlation with winter survival
To analyze the cause of functional differences depending on the haplotypes of LjFER and LjSRK, expression analysis was performed using 23 selected wild accessions. Results of gene expression analysis by real-time qPCR using the RNA samples collected from field growing accessions in five time points (August, October, December, February, and March) revealed a significant up-regulation of LjFER and LjSRK during winter time in the accessions with each winter-hardiness haplotypes. Moreover, the correlation between expression level in mid-winter (December and February) and winter-survival rate were detected in each gene. Thus the significance of winter-hardiness haplotypes of LjFER and LjSRK is considered to be their potential to induce the high expression in winter season. By the cold treatment test in the growth chamber, it was confirmed that the low temperature, not the photoperiod, triggered the up-regulation of LjFER and LjSRK in winter-hardiness haplotypes. Moreover, result from greenhouse experiment revealed that gene up-regulation of winter-hardiness haplotype of LjSRK basically detected during tested period, while up-regulation of winter-hardiness haplotype of LjFER observed in middle of the tested period with low temperature dependency. This observation resembled to the field time course analysis suggesting the different functional timing in contribution to winter-hardiness.

- Identification of co-expressed genes with haplotype dependent expressions of LjFER and LjSRK
To elucidate the haplotype dependent effect of LjFER and LjSRK in the regulatory network, co-expression analysis was carried out using the RNA-seq data set on field samples collected from summer to mid-winter. A total of 172 and 208 genes were identified as co-expressed gene with LjFER and LjSRK, respectively. Although none of the co-expressed genes of LjFER and LjSRK were overlapped, GO term enrichment analysis revealed that ten GO terms, including membrane activity and cell wall metabolism, were commonly enriched in co-expressed genes in both LjFER and LjSRK. In addition, genes related to lipid metabolism were co-expressed with LjSRK, while genes related to carbohydrate metabolism as well as components of ICE1-CBF pathway were clustered in LjFER. These results suggested that upregulation of LjFER and LjSRK in winter season long depending on the winter-hardiness haplotypes lead to the up-regulation of the different set of genes that contribute to enhance the physical toughness against harsh winter.

In this study, I revealed the novel insights of molecular mechanisms in local adaptation of L. japonicus to high latitude area with low temperature. By gaining winter-hardiness haplotypes in two receptor kinase genes, LjFER and LjSRK, up- regulation of the set of genes that provide protection against harsh winter including cell wall and cell membrane metabolism, lipid metabolism, carbohydrate metabolism, together with the activity of ICE1-CBF regulon were established during the winter season long. For perennial plant such as L. japonicus, the enhancement of tolerance in terms of surviving to the harsh of winter climate could have been critical for local adaptation to high latitude area. Moreover, the identified receptor kinase genes, LjFER and LjSRK may be considered as the master regulators of genes that contribute to the enhancement of winter-hardiness. Further analysis of these genes will contribute to the understanding of the environmental adaptation in the perennial plants. In addition, orthologues of LjFER and LjSRK will be good targets for improvement of temperature responses of economically important crops.

Conclusions
This study set out to elucidate the molecular mechanism in local adaptation of L. japonicus to high latitude area with low temperature. The results of this investigation show that by gaining winter-hardiness haplotype in two receptor kinase, LjFER and LjSRK bring the up-regulation of the set of genes that provide protection against harsh winter including cell wall and cell membrane metabolism, lipid metabolism, carbohydrate metabolism, together with the activity of ICE1-CBF regulon. For L. japonicus as a perennial plant, the enhancement of tolerance in terms of surviving to the harsh of winter climate could have been critical for local adaptation to high latitude area. By having the LjFER_Alt, LjSRK_Hap2/Hap3 could become an advantage for those accessions in northern population (subpopulation 3) for locally adapted, since by having these haplotypes could provide the protection against harsh winter season especially in high latitude region. The findings of this research provide the insight that LjFER and LjSRK could be considered as the master regulators of genes that contribute to the enhancement of winter-hardiness in perennial plant. Further analysis of these genes will contribute to the understanding of the environmental adaptation in the perennial plants as well as be a good target for improvement of temperature responses of economically important crops.