Specific haplotypes of the lipid-related OsGELP gene family are present in rice adapted to high latitudes

Specific haplotypes of the lipid-related OsGELP gene family are present in rice adapted to high latitudes | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 March 2025 V1 Latest version Share on Specific haplotypes of the lipid-related OsGELP gene family are present in rice adapted to high latitudes Authors : Kayyis Muayadah Lubba , Koichi Yamamori , and Yuji Kishima 0000-0002-0942-3371 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174175933.37603156/v1 330 views 180 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rice ( Oryza sativa ) originated in tropical regions and has adapted to higher latitudes; these adaptations affect photoperiod sensitivity and cold tolerance. Lipids are essential membrane components and function in sensing and responding to environmental conditions. Members of the lipid-related GDSL esterase/lipase (GELP) family have been implicated in stress responses. Here, to investigate the relationship between OsGELP genes and rice adaptation to high latitudes, we identified the haplotypes of each of the 115 OsGELP genes in 3000 rice accessions and sorted them based on latitude data and single-nucleotide polymorphisms, designating haplotypes present at an average latitude >35°N as high-latitude haplotypes (HLHs) and selecting 10 OsGELP genes with 11 HLHs for further analysis. Haplotype network and amino acid sequence analyses suggested that rapid changes involving a few amino acids encoded by genes with HLHs likely helped plants adapt to high latitudes. HLH-containing OsGELP s are often expressed in roots, suggesting that roots function in the adaptation of rice to high latitudes. Apart from OsGELP s, only 3 of the 14 known cold tolerance genes in rice have HLHs. We suggest that the 10 OsGELP genes with HLHs play crucial roles in the adaptation of rice to high latitudes. Specific haplotypes of the lipid-related OsGELP gene family are present in rice adapted to high latitudes Kayyis Muayadah Lubba 1 , Koichi Yamamori 2 , Yuji Kishima 3 * 1 Graduate School of Agriculture, Hokkaido University, 2 Graduate School of Agriculture, Kyoto University 3 Research Faculty of Agriculture, Hokkaido University *Corresponding author Yuji Kishima: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. [email protected] Funding This work was partly supported by grants from JSPS KAKENHI (No. 19H00937 and 23H02180 to Y. Ki) and RiceBACS in Science and Technology Research Partnership for Sustainable Development (SATREPS). Keywords: Adaptation, high-latitude haplotype, GDSL-type esterase/lipase, latitude, OsGELP, rice, root Summary statement: Rice accessions adapted to high latitudes contain OsGELP family genes with different haplotypes. This study reveals how these lipid metabolism–related genes have differentiated in high-latitude accessions. Abstract Rice ( Oryza sativa ) originated in tropical regions and has adapted to higher latitudes; these adaptations affect photoperiod sensitivity and cold tolerance. Lipids are essential membrane components and function in sensing and responding to environmental conditions. Members of the lipid-related GDSL esterase/lipase (GELP) family have been implicated in stress responses. Here, to investigate the relationship between OsGELP genes and rice adaptation to high latitudes, we identified the haplotypes of each of the 115 OsGELP genes in 3000 rice accessions and sorted them based on latitude data and single-nucleotide polymorphisms, designating haplotypes present at an average latitude >35°N as high-latitude haplotypes (HLHs) and selecting 10 OsGELP genes with 11 HLHs for further analysis. Haplotype network and amino acid sequence analyses suggested that rapid changes involving a few amino acids encoded by genes with HLHs likely helped plants adapt to high latitudes. HLH-containing OsGELP s are often expressed in roots, suggesting that roots function in the adaptation of rice to high latitudes. Apart from OsGELP s, only 3 of the 14 known cold tolerance genes in rice have HLHs. We suggest that the 10 OsGELP genes with HLHs play crucial roles in the adaptation of rice to high latitudes. Introduction Rice ( Oryza sativa ) originated in tropical and subtropical regions, but it can grow in a wide range of latitudes and is even cultivated at latitudes above 40°N (Gutaker et al., 2020). The adaptation of rice to different latitudes has been studied by examining flowering-related genes because latitude affects daylength(H. Gao et al., 2014; Guo et al., 2020). The adaptation of crops to new latitudes requires the retuning of flowering time to maximize yield potential. In potato ( Solanum tuberosum ), soybean ( Glycine max ), and maize ( Zea mays ), flowering genes were identified that improved adaptation to the limited summer growing season at higher latitudes during plant domestication (Huang et al., 2018; Kloosterman et al., 2013; Li et al., 2024; Maeda & Nakamichi, 2022). Unlike soybean, which originated in temperate regions and adapted to tropical regions, rice originated in tropical or southern regions and has adapted to northern regions, i.e., from low/middle to high latitudes (Kloosterman et al., 2013). To date, studies of the adaptation of rice to high latitudes have focused on flowering genes associated with ecotype (Izawa, 2007), and few other genes associated with this adaptation have been explored (Han et al., 2023). Therefore, more research is needed to understand the effects of latitudinal differences in temperature on rice. Lipids playi critical roles in signaling, energy storage, and membrane formation (Ursache et al., 2021). As lipids are essential membrane components in organs, tissues, and cells, they directly affect plant interactions with the environment (Sharma et al., 2023). The lipid-related Gly-Asp-Ser-Leu (GDSL) esterase/lipase proteins (GELPs) are essential enzymes in various physiological and developmental processes (Shen et al., 2022). These proteins have both esterase and lipase activities, function in the hydrolysis of ester bonds, and can act on lipids (Chepyshko, Lai, Huang, Liu, & Shaw, 2012). GELP proteins function in lipid metabolism, including the breakdown of lipids and the release of fatty acids (Shen et al., 2022). GELPs play key roles in plant responses to biotic and abiotic stress (Chepyshko et al., 2012). Rice contains 115 OsGELP genes that function in multiple processes, such as pollen exine development ( OsGELP110 and 115 ) (Huo et al., 2020; H. Zhang et al., 2020); cell wall formation ( OsGELP33 ) (B. Zhang et al., 2017) and OsGELP62 or DARX1 ) (L. Zhang et al., 2019); lipid homeostasis ( OsGELP34 ) (Yuan et al., 2020; H. Zhang et al., 2020; Zhao et al., 2020); disease resistance (M. Zhang et al., 2024); and secondary metabolism, such as pseudocholinesterase ( OsGELP91 ) and acetylcholinesterase ( OsGELP92 ) metabolism (Yamamoto & Momonoki, 2021). In addition, some GDSL LIPASE/ESTERASE genes are related to disease immunity ( OsGLIP1 , 2 , and 78 ) (Chen et al., 2016; M. Gao et al., 2017) and plant responses to water loss ( OsGELP112 or WDL1 ) (Park et al., 2010) and UV-B radiation ( OsGLIP1 )(He et al., 2021). Many GELP genes are closely associated with plant responses to abiotic stress (de Almeida, Barbosa, Ferraz, de Castro, & Ribeiro, 2024; Duan et al., 2023; Naranjo, Forment, Roldan, Serrano, & Vicente, 2006). However, the roles of GELP genes in environmental and regional adaptability in rice have not been investigated. The key to resolving this issue is to link the haplotypes of each gene to environmental and regional adaptability. In this study, we explored how rice adapts to a wide range of latitudes. Since lipids play crucial roles in various physiological processes in plants, including membrane structure, energy storage, and signaling, we focused on specific haplotypes of OsGELP genes in rice accessions grown in high-latitude regions and explored their roles in the adaptation of rice to these conditions. We also examined the organ-specific expression patterns of these genes to explore how different rice organs adapt to high latitudes. Our findings shed light on rice varieties with haplotypes of target OsGELP genes that have played a role in their adaptation, which could contribute to the development of region-specific rice varieties that thrive in harsh environmental conditions. Materials and Methods 2.1 Genome-wide identification of OsGELP genes in rice Among the 115 OsGELP genes in the rice genome, the chromosome positions of 113 OsGELP genes were obtained from RAP-DB (https://rapdb.dna.affrc.go.jp); the sequences of OsGELP36 and OsGELP73 were unavailable, as they were not present in RAP-DB ( Supplementary Data 1 ). All data, such as average latitude and haplotype ( Supplementary Data 2 ), subpopulation ( Supplementary Data 3 ), single-nucleotide polymorphisms (SNPs), and country of origin, were obtained from the IRRI SNP-seek Database (https://snpseek.irri.org) . Using the SNP-seek data, haplotypes were defined based on SNPs in each OsGELP gene sequence. General and specific analyses were performed. Global latitude analysis (GLA) used general latitude data or the location of the capital city in each country using the 3000 Rice Genomes (RG) data. Specific latitude analysis (SLA) used specific latitude data from the prefecture of origin of the Indonesian and Japanese accessions ( Figure S1 ). The genes were classified into those that were either latitude dependent or independent. The latitude-dependent genes were divided into two groups: genes with high-latitude haplotypes (HLHs), predominant at an average latitude >35°N, and genes lacking HLHs. Latitude-independent haplotypes were found in accessions originating at a latitude ranging from 0° to >40°N. High-latitude countries included China (39.9°N), Japan (35.70°N), Korea (37.55°N), the United States (38.9°N), and those in Europe (40°N to 60°N). A schematic representation of our methodology is shown in Figure 1 . 2.2 Extraction of haplotype and latitude data via GLA As described above, GLA was performed using general latitude data or the location of the capital city in each country using 3000 RG data. Haplotypes present in more than 30 accessions were considered for analysis (Angira, Cerioli, & Famoso, 2022). The average latitude was calculated based on the latitude of the capital city of the country where the haplotype was present. Latitude data were collected from the SNP-seek database. OsGELP genes containing HLHs and showing significant differences based on Duncan’s test ( p < 0.05) compared with another haplotype were selected during GLA. 2.3 Extraction of haplotype and latitude data via SLA As described above, SLA used the latitude of the prefecture of origin of each Indonesian and Japanese accession from the 3000 RG database to indicate the exact origins of the varieties. The latitude data were obtained from the Germplasm Catalogue 2010 from the Indonesian Ministry of Agriculture for the Indonesian accessions and the National Agriculture and Food Research Organization Genebank for the Japanese accessions. Seventy Indonesian accessions were used to represent low-latitude (11°S to 6°N) varieties, and 30 Japanese accessions were used to represent middle- to high-latitude (20°N to 45°N) varieties; these varieties are listed Supplementary Data 4 and 5 . Fisher’s exact test was also conducted during SLA to identify significant associations within the indica and japonica subpopulations. Genes containing HLHs from accessions grown at an average latitude above 35°N in the japonica subpopulation were selected for further analysis. 2.4 Extraction of haplotype data from Hokkaido varieties based on next-generation sequencing data Haplotype analysis of 22 varieties from Hokkaido (representing the northern part of Japan, with a latitude of 43°N) listed in Supplementary Data 6 was conducted on 10 selected OsGELP s (see Results). Whole-genome sequencing data for the 22 Hokkaido rice varieties are available from Fujino et al. (2021). The next-generation sequencing (NGS) data were mapped to the Nipponbare reference genome using Bowtie 2, and SNPs were called using GATK4 HaplotypeCaller. The resulting SNPs were further analyzed using TASSEL 5 and IGV software, with Nipponbare as the reference. The haplotype compositions of the 22 Hokkaido rice varieties were grouped based on SNPs obtained from NGS data, and the haplotypes and average latitudes were analyzed as described for the 3000 RG data. 2.5 Construction of a haplotype network The SNP-seek database was used to conduct haplotype analysis of genes containing HLHs to investigate the relationship between HLHs and other haplotypes in the network. The haplotype sequences were created by aligning SNPs using ClustalW in MEGA 11 (Tamura, Stecher, & Kumar, 2021). The haplotype network for each gene was constructed to analyze the genealogical relationships among the haplotypes using PopArt. Haplotype diversity was calculated with DnaSP software version 6 (Rozas et al., 2017). The rice regional accessions were sorted into 12 subpopulations: aro, aus, admix, ind1A, ind1B, ind2, ind3, indx, japx, subtrop, temp, and trop. In the SNP-seek Database, rice subpopulations aro, aus, and admix come from different regions, with aro being fragrant varieties from South Asia, aus being early-maturing and drought-tolerant from Bangladesh and Eastern India, and admix being hybrids found worldwide. indica subgroups (ind1A, ind1B, ind2, ind3, and indx) are mainly in South and Southeast Asia, with indx being mixed indica types. japonica admixed (japx) varieties combine japonica genetics and are found in East Asia and beyond. Subtropical japonica (subtrop) grows in mild climates, temperate japonica (temp) thrives in cooler regions with cold tolerance, and tropical japonica (trop) is suited to hot, humid areas with longer grains (SNP-seek database). 2.6 Phylogenetic analysis Multiple alignment (ClustalW) of the GDSL amino acid sequences in rice and other species was conducted using MEGA 11. Unrooted phylogenetic trees were built with the Neighbor Joining method. The phylogenetic tree was created using Poisson correction, pairwise deletion, and 1000 bootstrap replicates. 2.7 Gene expression profiling and protein structure analysis The gene expression profiles based on the normalized signal intensities (log 2 ) of 105 OsGELP genes were collected from the Rice Expression Profile Database (RiceXPro; https://ricexpro.dna.affrc.go.jp/); expression data for the 10 remaining OsGELP genes were not available ( OsGELP13 , 28 , 34 , 39 , 41 , 48 , 57 , 60 , 104 , and 115 ) ( Figures S10 and S11 ). Amino acid data were collected from the UniProt database (https://www.uniprot.org/), and the AlphaFold Protein Structure Database (AlphaFold DB; https://alphafold.ebi.ac.uk) was used for protein structure predictions and to collect the protein models. The models were constructed using AlphaFold3, an artificial intelligence algorithm developed by DeepMind. The 3D protein structure was visualized using ChimeraX (Meng et al., 2023). Results 3.1 Selection of OsGELP genes carrying HLHs We identified 115 potential OsGELP genes from both RAP-DB and the list assembled by Chepyshko et al. (2012). These genes are located across the 12 rice chromosomes, with several present in gene clusters ( Figure S2 , Supplementary Data 1 ). We used 115 OsGELP genes collected from the SNP-seek database of the 3000 RG data (https://snpseek.irri.org/snp.zul). All data, such as latitude, subpopulation, SNPs, and country of origin, were obtained from the SNP-seek database. Each of the 115 OsGELP genes had diverse haplotypes, with the number of haplotypes ranging from 2 to 17 and an average of approximately 5.8 haplotypes per gene. Haplotypes present in more than 30 accessions were considered for further analysis (Angira et al., 2022). We considered China (39.9°N), Japan (35.70°N), Korea (37.55°N), the United States (38.9°N), and European countries (40°N to 60°N) to be high-latitude countries. Many haplotypes of the OsGELP genes were detected under specific geographical conditions characterized by different average latitudes ( Supplementary Data 2 ). For GLA, the average latitude was calculated based on the latitude of the capital city of the country where the haplotype was present. The average latitudes of the haplotypes of all OsGELP genes ranged from 6.58°N to 37.16°N ( Supplementary Data 2 ). Comparing the distribution between genes with or without HLHs in the 3000 RG data, HLHs were found in varieties in high-latitude regions. OsGELP74 lacks an HLH; its haplotypes are found in latitudes from 15.13°N to 25.23°N, representing the optimal latitude for rice growth ( Figures S3A and S3C ). All haplotypes of OsGELP74 are widely and randomly distributed in the 3000 RG data across the latitudes. Conversely, OsGELP65 has two HLHs, with different haplotypes showing different distributions ( Figures S3B and S3D ). Varieties carrying either HLH hap_184 or hap_369 in OsGELP65 are present almost exclusively in high-latitude regions ( Figures S3B and S3D ), indicating that both HLHs are specific high-latitude genotypes that might be responsible for adaptation to high latitudes. Among the 115 OsGELP s, 12 genes had haplotypes predominant at latitudes above 35°N: OsGELP4 , 18 , 19 , 42 , 58 , 60 , 64 , 65 , 66 , 90 , 104 , and 107 ( Figure 2, Table S1 ). 3.2 Selection of HLHs for SLA A limitation of GLA is that the exact latitudes of the rice varieties were not reflected by the latitudes of the capital cities in countries with a wide range of latitudes from north to south, such as Japan, China, and the United States. To make the latitude data for haplotypes as accurate as possible for the place of origin where a variety was created, we conducted SLA using specific latitude data for two countries: Indonesia and Japan. For SLA, we employed 70 Indonesian and 30 Japanese accessions. None of the 70 Indonesian accessions contained HLHs of OsGELP genes or latitude-specific haplotypes, whereas the 30 Japanese accessions had HLHs for most of the 12 genes selected by GLA ( Figure 2, Supplementary Data 4 and 5 ). Specifically, SLA revealed that in the Japanese accessions, OsGELP18, 19, 42, 58, 60, 64, 66, 90 , and 107 each had one HLH, and OsGELP65 had two HLHs: hap_184 and hap_369 ( Figure 3 ). SLA identified 11 HLHs for the 10 genes that were also identified by GLA (hereafter “selected genes”), but two genes, OsGELP4 and OsGELP104 , did not have HLHs in the Japanese accessions ( Figure 3A and 3K, Table S1 ). 3.3 Confirmation of HLHs in high-latitude accessions Owing to its wide range of latitudes (22°N to 43°N), Japan has a rich variety of rice accessions in each prefecture. To assess whether HLHs are primarily found in the northern part of Japan, we focused on Hokkaido, which has the lowest ambient temperatures in Japan. We examined the distributions of HLHs of 10 selected OsGELP genes using 22 Hokkaido varieties. Each variety carried HLHs for 4 to 9 of the 10 OsGELP genes. Among the 10 genes with HLHs, HLHs of OsGELP42 and OsGELP58 were found in 50% of the 22 Hokkaido varieties, representing the lowest proportion. By contrast, the HLH of OsGELP60 was present in all 22 varieties ( Figure 4 ). These HLHs of the 10 selected genes were confirmed to be present only in japonica and temperate japonica rice subpopulations but not in indica ( Figure S4 ). Fisher’s test validated that the HLHs were significant components in the japonica and temperate japonica rice subpopulations. These results suggest that these haplotypes contribute to the adaptation of high-latitude rice varieties to northern regions. 3.4 Haplotype networks of genes with HLHs GLA and SLA selected 11 HLHs for 10 genes: OsGELP18 , 19 , 42 , 58 , 60 , 64 , 65 , 66 , 90 , and 107 ( Figure 3 ). We created haplotype networks for each of these genes to illustrate the relationships among haplotypes, their origins, and the population size of each haplotype ( Figure 5 ). The large circles represent haplotypes found primarily at mid-latitudes, averaging between 18°N and 25°N. HLHs for seven of the selected genes ( OsGELP18 , 19 , 42 , 58 , 64 , 65 , and 90 ) were directly derived from the major haplotype ( Figures 5A, B, C, D, F, G, and I ), while the remaining three genes ( OsGELP60 , 66 , and 107 ) had HLHs branching from minor haplotypes of the network ( Figures 5E, H, and J ). The genetic variations of these HLHs could have facilitated adaptation to different environmental conditions at specific latitudes. Among the proteins encoded by the first seven genes with HLHs, a few amino acid changes might have occurred that rapidly enhanced adaptation to high latitudes. OsGELP65 has two HLHs, hap_184 and hap_369, which are closely positioned in the haplotype network ( Figure 5G ). This proximity points to the recent diversification and common evolutionary origin of these haplotypes ( Figure 5 ). Their proximity suggests that they either provide similar functional benefits or were selected under comparable environmental pressures, which might be related to the functions of GELPs in plants. 3.5 Phylogenetic analysis and amino acid changes related to haplotypes We performed phylogenetic analysis of GELP genes in rice and other plant species. The OsGELP genes clustered with other GDSL genes from diverse plant species, suggesting that they might be evolutionarily conserved and play similar roles among plants. Neighboring genes from other species (e.g., AtFXG1 from Arabidopsis thaliana , ZmAChE from maize, and BnSCE3 from Brassica napus ) form distinct clades, reflecting functional or evolutionary divergence ( Figure S5 ). Among the 10 selected genes, 4 pairs of OsGELP genes ( OsGELP18 and 19 , OsGELP42 and 90 , OsGELP64 and 65 , and OsGELP60 and 66 ) were identified as paralogs ( Figure S5 ). We analyzed amino acid changes resulting from SNP variations in each haplotype of these paralogous genes. As shown in Figures 6 , the mutations observed between the major haplotype sequences and the HLHs have occurred in diverse amino acid positions. For example, the amino acid changes between OsGELP19 and OsGELP18 occurred in different motifs ( Figure S6 ). Despite these mutations, we identified two conserved motifs across the 10 selected genes ( Figure S6 ). Mutations or amino acid changes within these genes often occurred in distinct motifs or positions, suggesting that the mutation independently occurred in each HLH. Notably, SNPs between the major haplotypes and HLHs resulted in amino acid substitutions and a decrease in hydrogen (H)-bond stability. This is illustrated by the paralogous genes OsGELP18 and OsGELP19 , which were found to be homologous based on their encoded amino acid sequences ( Figures 6A and 6B ). Haplotype analysis of 22 Hokkaido rice varieties revealed similarities between these two genes, as shown in the haplotype grouping. The rice varieties Akage, Hayayuki, Hokkaido, Nourin 15, and Wasefukoku exhibited haplotypes distinct from the 17 other varieties. This was further confirmed by the presence of a 2-bp insertion in OsGELP18 at genomic position Chr. 1 (26200736–26200737) and a 10-bp insertion in OsGELP19 at genomic position Chr. 1 (26203718–26203727) ( Figures 6A and 6B ). Changes in SNPs in the exons of OsGELP18 and OsGELP19 led to single amino acid substitutions. In OsGELP18, the amino acid glutamic acid (E) in hap_7 and hap_3 was replaced with lysine (K) in HLH (hap_26). Similarly, in OsGELP19 , phenylalanine (F) in hap_7 and hap_811 was replaced with leucine (L) in HLH (hap_646). These single amino acid changes are expected to result in altered H-bonding ( Figure 6 ). In addition to haplotypes encoding single amino acid substitutions, OsGELP64 encodes a protein with five amino acid differences across six haplotypes. For example, HLH (hap_200) encodes a protein in which amino acid position 255 contains alanine (A), whereas it contains valine (V) in proteins encoded by other haplotypes. This change occurred within the alpha-helix region ( Figures 6C and D ). Mutations in alpha-helical regions tend to be more robust than those in beta-strands, as helices tolerate more sequence variation without disrupting secondary structure. This robustness is primarily due to the higher number of interacting residues in helices than in strands or coil regions (Abrusan & Marsh, 2016). Furthermore, OsGELP58 ( Figure S7 ) and OsGELP66 ( Figure S8 ) exhibited amino acid changes resulting in differences in H-bonding between the major haplotypes and HLH. We also observed SNP changes in intron regions in genes such as OsGELP65 ( Figure S9 ). 3.6 Organ-specific OsGELP gene expression We classified 105 OsGELP genes based on their organ-specific expression patterns ( Supplementary Data 7 ) by constructing a heatmap of their normalized relative gene expression levels (normalized signal intensity [log 2 ]) in leaf, root, stem, and anther tissue (data were not available for OsGELP13 , 28 , 36 , 39 , 41 , 48 , 57 , 60 , 104 , or 115 ) ( Figures S10 and S11 ). We generated the gene expression profiles from RiceXPro data using a single microarray platform with probes based on manually curated gene models in RAP-DP and full-length rice cDNA sequence information in the KOME database. The 105 OsGELP genes showed various levels of expression in leaf blades, leaf sheaths, roots, stems, and anthers ( Supplementary Data 7 ). Figure 7A shows the normalized relative expression levels of the nine selected OsGELP genes in various rice organs, including the leaf blade, leaf sheath, root, stem, inflorescence, anther, pistil, palea and lemma, ovary, embryo, and endosperm. We detected significant differences in expression among organs based on the average expression level in each organ, as determined by Duncan’s test ( Figure 7A ). Overall, these genes were expressed at the highest levels in roots and stems, with several genes showing peak expression in these organs. OsGELP18 and OsGELP19 showed notably high expression in roots, while OsGELP58 and OsGELP66 exhibited elevated expression in stems. In Figure 7B , the organ with the highest expression level for each gene is highlighted in blue. For instance, OsGELP18 was expressed at the highest levels in roots, while OsGELP19 expression peaked in stems. These patterns suggest that OsGELP genes might have specialized functions depending on the organ in which they are most highly expressed. The overall expression profiles point to functional specialization of OsGELP genes in rice. The roots and stems were the primary sites of expression for many of these genes, suggesting they might function in structural support, nutrient uptake, or other root- and stem-specific processes. These results indicate which organs might require adaptation at high latitudes. At the same time, these genes might be indispensable for the development of the corresponding organs. Genes with HLHs that are expressed in roots and stems might strongly contribute to the adaptation of rice to high latitudes. Discussion 4.1 Haplotype diversity of OsGELP genes at different latitudes We identified 11 HLHs for 10 OsGELP genes: OsGELP18 , 19 , 42 , 58 , 60 , 64 , 65 , 66 , 90 , and 107 . Genes with latitude-dependent haplotypes are known to be present in rice (F. Zhang et al., 2021), but no previous study had identified such a large number of genes related to high-latitude adaptation in the same gene family. These genes might play significant roles in the adaptation of rice to high-latitude environments. The temperate japonica subpopulation has high frequencies of favorable haplotypes or specific HLHs for OsGELP genes, suggesting that these temperate japonica varieties share metabolic similarities that contribute to adaptation to high-latitude environments. GELP s are key genes involved in the metabolism and degradation of lipids in the epidermis in many plant organs (de Almeida et al., 2024). The differentiation of GELP genes is thought to be important for creating plant plasticity in response to environmental cues and stress. The functions of 12 GELP genes in the rice genome remain to be studied ( Table 1 ). Here, we searched for specific haplotypes of each OsGELP gene in rice accessions adapted to high-latitude regions and obtained evidence that members of the OsGELP family have important functions related to environmental adaptation and stress responses in rice at high latitudes. To assess the correlation between haplotype classifications and latitude, we analyzed specific accessions from Indonesia and Japan. Indonesian accessions originated in low- or middle-latitude regions, whereas Japanese accessions originated in high-latitude regions. The unique haplotypes in accessions distributed in high-latitude regions are likely beneficial alleles in stressful environments (F. Zhang et al., 2021). In this study, we selected rice varieties from Hokkaido (northern Far East) as materials adapted to high latitudes because they are characterized by photoperiod insensitivity and cold tolerance (Fujino, Nishimura, Kiuchi, Hirayama, & Sato, 2017). Hokkaido rice varieties differ from other japonica varieties in that more than 50% of the 10 OsGELP genes have HLHs ( Figure 4 ). At least 10 OsGELP genes are associated with high-latitude adaptation, and their HLHs are present in high-latitude varieties. To our knowledge, no other gene families with 11 or more high-latitude-specific haplotypes have been identified in the rice genome. These results indicate that OsGELP genes have significantly contributed to rice cultivation in the northern Far East. 4.2 Genes with HLHs are preferentially expressed in roots and stems Four out of the 10 OsGELP genes with HLHs were most highly expressed in roots, followed by 2 that were most highly expressed in stems. Roots and stems are in direct contact with the ambient temperature and soil and are considered primary sensors of various abiotic stress factors (Zhou, Sommer, & Hochholdinger, 2021). The stem is the organ most exposed to the outside air and most susceptible to environmental influences. Therefore, the stem must have contributed greatly to plant adaptation to the environment. Surprisingly, however, few studies have focused on the mechanism by which the stem adapts to the environment. The metabolism of lipids in the stem might play an important role in the adaptation of rice to high latitudes. Unlike wheat ( Triticum aestivum ), which is native to temperate zones, temperatures below 20°C pose a major abiotic constraint to rice, which is native to the tropics and subtropics. Cold stress is detrimental to the development and functions of rice roots and stems (Sanchez-Bermudez, Del Pozo, & Pernas, 2022). When cold-sensitive rice lines are exposed to cold stress, their root hairs are shortened and reduced in number, resulting in an overall reduction in root dry weight (Rativa et al., 2020). By contrast, cold-tolerant lines do not suffer as much damage in response to cold stress. Lipid-related compounds are concentrated and accumulate in roots, strongly suggesting a link between abiotic stress and lipids in plants. In wheat, the overall accumulation profiles of lipid compounds differ between cold-tolerant and -sensitive lines when cold stress is applied (Cheong et al., 2022). Cold stress inhibits root development in cold-sensitive rice lines by promoting the accumulation of reactive oxygen species in roots (relative to the levels in cold-tolerant rice) via processes such as lipid peroxidation (Rativa et al., 2020). In this study, we also showed that OsGELP genes are mainly expressed in roots ( Figure 7A ). Four of these genes, OsGELP18 , 64 , 65 , and 107 , have HLHs ( Figure 7B ). OsGELP64 and 65 are highly homologous to the Alopecurus myosuroides gene AmGDSH1 , which encodes a carboxylesterase enzyme involved in the hydrolysis of herbicides, particularly aryloxyphenoxypropionate, in roots (Gershater, Cummins, & Edwards, 2007). Fewer OsGELP genes are expressed in other tissues than in roots, and these genes also have fewer HLHs. Rice roots have also likely evolved a group of lipid-related genes that can resist cold stress, thus enabling their growth at high latitudes. This is consistent with the idea that the adaptation of rice to high latitudes was achieved through enhanced cold tolerance in roots. All vascular plants are protected from the environment by a cuticle; this lipophilic layer, which is synthesized by epidermal cells, is composed of a cutin polymer matrix and waxes (Suh et al., 2005). The waxy, protective cuticle coats all primary aerial plant tissues, including stems. The synthesis of the cuticle requires the extensive export of lipids from epidermal cells to the plant surface (Pighin et al., 2004). Lipids also contribute to stem strength by forming suberin and lignin (J. Tang et al., 2020; Y. Tang, Lu, Sheng, Zhao, & Tao, 2023), which fortify vascular tissues, aid in water and nutrient transport, and support stress responses (Pighin et al., 2004). Members of the GELP family catalyze the polymerization of cutin, and candidate proteins have been proposed that mediate interactions between cutin or suberin and other cell wall components (Philippe et al., 2020). 4.3 Importance of the major HLHs of OsGELP s for adaptation to high-latitude regions Three out of 14 genes reported to be associated with cold tolerance in rice at the booting or seedling stage had HLHs: OsMPK3 , LTG1 , and OsAPX1 ( Table 2, Figures S12 and S13 ) . We also looked for HLHs of flowering-related genes. Of the seven flowering-related genes examined, only two ( Ghd7 and DHT8 ) had HLHs ( Table 3, Figure S14 ). Some haplotypes were detected at latitudes above 25°N, but not above 35°N, which we set as a threshold for the high-latitude OsLEA9, OsMKK70, CTB2, CTB4a, OsMYB4, Ctb1, qCTB7, bZIP73, LTT1 , and HAN1. These results indicate that the 10 OsGELP s and other genes with HLHs are associated with the adaptation of rice to high-latitude conditions, rather than cold tolerance and flowering-related genes being the only ones responsible for high-latitude adaptation. The OsGELP genes might play crucial roles in adaptation to environmental stress, including conditions found in high-latitude regions, where cold stress can be more severe. Most genes related to cold tolerance in rice identified to date are not known to be associated with lipid metabolism, although lipid metabolism and membrane lipids play vital roles in abiotic and biotic stress responses in plants (Gusain, Joshi, & Joshi, 2023; Moellering, Muthan, & Benning, 2010). Based on our findings, all 10 OsGELP genes analyzed contain diverse mutations or amino acid changes between the major haplotype and HLH, pointing to the functional independence of each gene. In addition, changes in H-bonds are important for stable lipid metabolism (Bondar & White, 2012). Indeed, we determined that some OsGELPs have amino acid changes that affect the number of H-bonds. H-bonds stabilize protein–lipid interactions, particularly those in membrane-bound enzymes related to lipid metabolism (Pogozheva, Tristram-Nagle, Mosberg, & Lomize, 2013). The relationship between lipids and latitude in rice affects how plants adapt their lipid compositions in response to varying environmental conditions associated with latitude, such as temperature and light intensity. In general, latitude influences temperature and light conditions, which in turn affect lipid metabolism in rice. Rice grown at higher latitudes is often exposed to cooler temperatures. To maintain membrane fluidity under colder conditions, the levels of unsaturated fatty acids in lipids tend to increase in rice. 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Workflow of global and specific latitude analyses to identify 115 OsGELP genes related to latitude across 3000 rice genomes. Figure 2. GLA of selected OsGELP genes. A. OsGELP4 ; B. OsGELP18 ; C. OsGELP19 ; D. OsGELP42 ; E. OsGELP58 ; F. OsGELP60 ; G. OsGELP64 ; H. OsGELP65 ; I. OsGELP66 ; J. OsGELP90 ; K. OsGELP104 ; L. OsGELP107 . Blue dots, Japanese accessions; red dots, Indonesian accessions. Different letters indicate significant differences based on Duncan’s test ( p < 0.05). Haplotype identified in more than 30 accessions from the 3000 rice genomes were considered for analysis. Figure 3. SLA of selected OsGELP genes. A. OsGELP4 ; B. OsGELP18 ; C. OsGELP19 ; D. OsGELP42 ; E. OsGELP58 ; F. OsGELP60 ; G. OsGELP64 ; H. OsGELP65 ; I. OsGELP66 ; J. OsGELP90; K. OsGELP104 ; L. OsGELP107 . Blue dots, Japanese accessions; red dots, Indonesian accessions. The y axis show the latitude average in each haplotypes. Different letters indicate significant differences based on Duncan’s test ( p < 0.05). Haplotypes identified in more than 30 accessions from the 3000 rice genomes were considered for analysis. Figure 4. Distribution of the 10 selected HLH genes in Hokkaido rice varieties. Gray boxes indicate HLHs, and numbers on the x - and y -axes indicate the total number of HLHs. Figure 5. Haplotype networks of selected OsGELP genes. A. OsGELP18 ; B. OsGELP19 ; C. OsGELP42 ; D. OsGELP58 ; E. OsGELP60 ; F. OsGELP64 ; G. OsGELP65 ; H. OsGELP66 ; I. OsGELP90 , and J. OsGELP107. Haplotypes are represented by circles; the size of each circle reflects the frequency of the corresponding haplotype. Lines connecting branches represent single mutational events. Figure 6. SNPs, haplotype variations, and the resulting amino acid changes for selected genes. A. SNPs and haplotype variation of OsGELP18 . B. SNPs and haplotype variation of OsGELP19. OsGELP18 and 19 have HLHs present in 77% of Hokkaido varieties. C. SNPs and haplotype variation of OsGELP64 , which has an HLH present in 85% of Hokkaido varieties. The yellow rows in A – C show the varieties with the HLH. D. The amino acid changes encoded by each haplotype of OsGELP64. Red arrows indicate amino acid changes in the alpha helix. E. Protein structures, including alpha-helix (α) (spiral) and beta-sheet (β) (green) positions, encoded by hap_81 and hap_200 (HLH) of OsGELP64. Red arrows, main haplotype; blue arrows, HLH. The numbers in parentheses show the average latitudes of accessions with the haplotype. Figure 7. Normalized relative expression levels of the 10 selected OsGELP genes based on the RiceXPro database. A. Normalized relative expression levels of each selected gene in leaf blades, leaf sheaths, roots, stems, inflorescences, anthers, pistils, palea and lemma, ovaries, embryos, and endosperm. Asterisks indicate significant differences in average expression levels (red bar) in each organ based on Duncan’s t -test (* p < 0.05). B. Normalized relative expression levels of each gene in several organs; blue indicates the highest expression level of each gene among the 12 organs examined. Table 1 . GELP genes whose functions were identified in previous studies Table 2. Classes of known cold tolerance genes based on the presence or absence of HLHs Table 3. Classes of known photoperiodic flowering genes based on the presence or absence of HLHs Supplementary Material File (lubba et al. pce figure (7) 2025.3.9.pptx) Download 2.36 MB Information & Authors Information Version history V1 Version 1 12 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords adaptation development high-latitude haplotype osgelp rice Authors Affiliations Kayyis Muayadah Lubba Hokkaido Daigaku Nogakubu Daigakuin Nogaku Kenkyuin Nogakuin View all articles by this author Koichi Yamamori Kyoto Daigaku Nogaku Kenkyuka Nogakubu View all articles by this author Yuji Kishima 0000-0002-0942-3371 [email protected] Hokkaido Daigaku Nogakubu Daigakuin Nogaku Kenkyuin Nogakuin View all articles by this author Metrics & Citations Metrics Article Usage 330 views 180 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Kayyis Muayadah Lubba, Koichi Yamamori, Yuji Kishima. Specific haplotypes of the lipid-related OsGELP gene family are present in rice adapted to high latitudes. Authorea . 12 March 2025. DOI: https://doi.org/10.22541/au.174175933.37603156/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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