Suppressing an auxin efflux transporter enhances rice adaptation to temperate habitats

Cui, Yanchun; Huang, Lifang; Liu, Peng; Wang, Xiaodong; Wu, Bi; Tan, Yongjun; Huang, Xuan; Hu, Xiaojie; He, Zhankun; Xia, Yuqi; Li, Zebang; Zhang, Wenli; Tang, Wenbang; Xing, Yongzhong; Chen, Caiyan; Mao, Donghai

doi:10.1038/s41467-025-59449-z

Download PDF

Article
Open access
Published: 02 May 2025

Suppressing an auxin efflux transporter enhances rice adaptation to temperate habitats

Yanchun Cui¹^na1,
Lifang Huang¹^na1,
Peng Liu¹^na1,
Xiaodong Wang^1,2^na1,
Bi Wu ORCID: orcid.org/0000-0002-1151-1848³,
Yongjun Tan ORCID: orcid.org/0000-0003-1041-4912⁴,
Xuan Huang^1,2,
Xiaojie Hu^1,5,
Zhankun He¹,
Yuqi Xia^1,2,
Zebang Li^1,2,
Wenli Zhang ORCID: orcid.org/0000-0003-0710-1966⁶,
Wenbang Tang⁴,
Yongzhong Xing³,
Caiyan Chen ORCID: orcid.org/0000-0001-7704-4691^1,2 &
…
Donghai Mao ORCID: orcid.org/0000-0002-9850-149X^1,2,7

Nature Communications volume 16, Article number: 4100 (2025) Cite this article

3422 Accesses
26 Altmetric
Metrics details

Subjects

Abstract

Rice (Oryza sativa L.), a chilling-sensitive staple crop originating from tropical and subtropical Asia, can be cultivated in temperate regions through the introduction of chilling tolerance traits. However, the molecular mechanisms underlying this adaptation remain largely unknown. Herein, we show that HAN2, a quantitative trait locus, confers chilling tolerance in temperate japonica rice. HAN2 encodes an auxin efflux transporter (OsABCB5) and negatively regulates chilling tolerance, potentially via auxin-mediated signaling pathway. During rice domestication, HAN2 has undergone selective divergence between the indica and temperate japonica subspecies. In temperate japonica rice, the insertion of a Copia long terminal repeat retrotransposon downstream of HAN2 reduces its expression, thereby enhancing chilling tolerance and facilitating adaptation to temperate climates. Introgression of the temperate japonica HAN2 allele into indica rice significantly improves chilling tolerance at both seedling and booting stages. These findings advance our understanding of rice northward expansion and provide a valuable genetic resource for improving yield stability under chilling stress.

Natural variation of CTB5 confers cold adaptation in plateau japonica rice

Article Open access 25 January 2025

Genome-wide association study and gene network analyses reveal potential candidate genes for high night temperature tolerance in rice

Article Open access 24 March 2021

Molecular mapping and characterization of QTLs for grain quality traits in a RIL population of US rice under high nighttime temperature stress

Article Open access 25 March 2023

Introduction

Chilling stress is a critical environmental stress factor that restricts crop growth and geographical distribution, thereby impacting crop yield. Rice, a staple food for over 50% of the global population, originated in tropical and subtropical regions and is inherently sensitivity to chilling stress¹. Some rice ecotypes, such as whole indica rice and part of tropical japonica rice, which predominantly grow in tropical and subtropical areas, are particularly sensitive to chilling¹. Conversely, temperate japonica rice acquired chilling tolerance and adapted to temperate regions during domestication¹. Chilling tolerance in rice is a complex trait governed by quantitative trait loci (QTL). To date, many QTLs associated with the difference in chilling tolerance between indica and japonica rice have been primarily mapped across 12 rice chromosomes through linkage mapping or genome-wide association studies². However, only a few of these QTLs have been identified and characterized, including LTG1³, COLD1⁴, qCTS-9⁵, LGS1⁶, qCT7⁷, HAN1¹, COLD11⁸, COG1-4^9,10,11,12, and COLD6¹³ for seedling tolerance; CTB1¹⁴, CTB4a¹⁵, bZIP73¹⁶, CTB3¹⁷, CTB5¹⁸, and CTB6¹⁹ for booting tolerance; qLTG3-1²⁰ and OsUBC12²¹ for germination tolerance. To develop climate-resilient rice varieties, it is essential to clone more chilling tolerance QTLs from rice.

Plants have evolved various strategies to rapidly sense, respond and adapt to chilling stress via calcium signalling, MAPK cascades, and transcription factors. The potential chilling sensors COLD1 trigger calcium (Ca²⁺) signalling to confer chilling tolerance in cells, while a chilling sensor complex of COLD6 and osmotin-like 1 (OSM1) triggers 2’, 3’-cyclic adenosine monophosphate (2’, 3’-cAMP) production to enhance chilling tolerance in rice^4,13. These chilling-induced second messenger signals can be sensed by MAPK cascades, which transduce the low-temperature signals downstream via phosphorylation pathways, thereby facilitating the adaptive responses to chilling stress. Among them, OsMAP1/OsMAPK3 is a gene that encodes a mitogen-activated protein kinase and plays a crucial role in regulating chilling tolerance during both the seedling and flowering stages^22,23. Calcium-dependent protein kinase can direct phosphorylate and activate OsMAP1/OsMAPK3 to improve chilling tolerance in rice²⁴. Chilling signals can also be transited by MAPK cascades, such as OsMKK6-OsMPK3, to enhance chilling tolerance in rice²⁵. OsMAP1/OsMAPK3 phosphorylates and stabilises OsbHLH002, which targets OsTPP1 to increase its transcription, leading to increased trehalose level and enhanced chilling tolerance in rice²². In addition to OsMAP1/OsMAPK3, chilling signalling is transited by many transcription factors, such as OsMADS57²⁶, OsTB1²⁶, OsWRKY94²⁶, MYBS3²⁷, OsGRF6²⁸, and OsWRKY76²⁹ to balance the tolerance and growth. However, it remains unreported whether the pivotal gene OsMAP1/OsMAPK3, which regulates chilling tolerance concurrently at the seedling and booting stages, is subject to regulation by transcription factors.

Plant hormones play a crucial role in regulating the chilling tolerance of rice. Hormones such as jasmonic acid (JA), abscisic acid (ABA), and ethylene are instrumental in enabling rice to adapt to chilling environments by modulating physiological and molecular mechanisms³⁰. HAN1 encodes a monooxygenase from the cytochrome P450 family that facilitates the conversion of active jasmonoyl-L-isoleucine to inactive 12-hydroxy-jasmonoyl-L-isoleucine. This process regulates JA-mediated responses to chilling stress and enhances chilling tolerance in rice¹. bZIP73^Jap interacts with bZIP71 to modulate ABA levels and maintain a balance of reactive oxygen species, consequently enhancing rice’s resilience to chilling stress¹⁶. OsUBC12 encodes an E2 ubiquitin-conjugating enzyme that negatively regulates ABA signalling²¹. Auxin plays a crucial role in plant growth, development, and environmental adaptation. A previous study reported the impact of auxin on the adaptability of plants to chilling stress³¹; however, the exact mechanism by which auxin regulates chilling tolerance is still not well understood. Moreover, no studies have reported on the involvement of auxin in the formation of chilling tolerance traits in temperate japonica rice during domestication.

In this study, we isolate and functionally characterize one of the QTL genes, which is named HAN2 (“HAN” means “chilling” in Chinese). HAN2 encodes an ABCB transporter (OsABCB5) with auxin efflux function. The gene negatively regulates chilling tolerance in rice, potentially via auxin-mediated signalling pathway. Introgression of the temperate japonica rice allele of HAN2, which contains a Copia long terminal repeat retrotransposon (Copia LTR) insertion downstream, not only enhances chilling tolerance at both seedling and booting stages but also demonstrates its potential for improving yield stability and supporting climate-resilient rice breeding.

Results

HAN2 negatively regulates chilling tolerance in rice

Temperate japonica rice typically exhibits stronger chilling tolerance than indica rice (Supplementary Fig. 1a-b). The genetic bases of chilling tolerance were investigated using a set of chromosome segment substitution lines derived from a backcross between the temperate japonica donor cultivar Nipponbare (NPB) and the indica recipient cultivar 93-11. The results showed that the chilling tolerance of NIL-HAN2^NPB was approximately twice that of 93-11 (Fig. 1a, b). Genome-wide genotyping revealed a substituted segment spanning the 26.0–29.9 Mb interval on chromosome 1 (Supplementary Fig. 2). This suggests the presence of a QTL in this region, named HAN2, which confers chilling tolerance in rice, consistent with previously reported findings³².

To isolate the causal QTL gene underlying HAN2, NIL-HAN2^NPB was further backcrossed to 93-11 for high-resolution mapping, generating approximately 3200 BC₁F₂ progeny plants. Based on the chilling tolerance performance of seedlings among these progeny lines, HAN2 was mapped to the interval flanked by markers M4 (R1ID2876) and M5 (R1ID2879) and further narrowed to a 30.0 kb genomic region containing three predicted genes (Fig. 1c, Supplementary Fig. 3a–f and Supplementary Table 1). Within this 30.0 kb interval, LOC_Os01g50090 was identified as a retrotransposon. Differential expression analysis revealed that the auxin transporter gene OsABCB5 (LOC_Os01g50100) was the only gene exhibiting significant differential expression in seedlings shoot between the two parental lines (Fig. 1d and Supplementary Fig. 4). Additionally, LOC_Os01 g50100 expression changed markedly in response to chilling stress (Supplementary Fig. 5a), and its expression level was significantly higher in indica rice than temperate japonica rice (Supplementary Fig. 5b). Based on these findings, we identified OsABCB5 as the candidate gene for HAN2.

To validate whether OsABCB5 is HAN2, knockout and overexpression lines were developed in the background of temperature japonica rice cultivar ZH11. In the CRISPR/Cas9-derived knockout lines han2-1 and han2-2, chilling tolerance was significantly enhanced at the seedling stage (Fig. 1e, f and Supplementary Fig. 6a), whereas chilling tolerance was reduced in the overexpression lines (Fig. 1g, h and Supplementary Fig. 6b). Introduction of the pHAN2::HAN2^93-11-GUS expression vector into NIL-HAN2^NPB resulted in transgenic lines with elevated HAN2 expression and decreased chilling tolerance (Supplementary Fig. 7a–c). These results confirm that OsABCB5 corresponds to HAN2, which negatively regulates chilling tolerance in rice.

HAN2 functions as an auxin efflux transporter

HAN2 was predicted to encode the ABCB transporter OsABCB5, which may be involved in auxin transport (Supplementary Fig. 8a, b). To validate the role of HAN2/OsABCB5 in auxin transport, we conducted a series of transport activity assays. First, we performed a deuterium-labelled auxin 3-indoleacetic acid-d₅ (d₅-IAA) transport assay in yeast cells. No significant difference in d₅-IAA content was observed between HAN2-GFP and GFP-expressing yeast cells after 3 h of incubation with 150 μM d₅-IAA (Supplementary Fig. 9a, b). However, after 12 h of efflux, d₅-IAA content was significantly lower in HAN2-GFP-expressing cells than in the GFP controls (Fig. 2a, b). Second, in Xenopus oocytes, no significant difference in d₅-IAA content was observed between GFP-HAN2 and GFP-expressing oocytes after 4 h of incubation with 150 μM d₅-IAA (Supplementary Fig. 9c). However, following 4 h of efflux, GFP-HAN2-expressing oocytes exhibited a more pronounced reduction in d₅-IAA levels compared with GFP-expressing oocytes (Fig. 2c, d). Additionally, a d₅-IAA transport activity assay in rice protoplasts showed that wild type (WT) protoplasts had a significantly higher relative transfer of d₅-IAA efflux than han2 protoplasts at 1 h, and 2 h (Fig. 2e). These results demonstrate that HAN2 functions as an auxin efflux transporter.

**Fig. 2: Functional analysis of HAN2 in auxin efflux transport.**

The expression pattern of HAN2 was examined to understand its functional role. RT-qPCR analysis showed that HAN2 was primarily expressed in the leaf, with particularly high expression in mature leaf (Supplementary Fig. 10a, b). This result was further validated by GUS histochemical staining in pHAN2::HAN2^93-11-GUS transgenic lines (Supplementary Fig. 10c). Immunofluorescence analysis using an anti-GUS antibody in these transgenic lines revealed that HAN2 is localized in the vascular bundle sheath parenchyma cells of the leaf (Supplementary Fig. 10d). Additionally, subcellar localization assays demonstrated that HAN2 resides in the plasma membrane of rice protoplasts (Supplementary Fig. 10e). Together, these findings indicate that HAN2 functions as an auxin transporter in the xylem of mature leaves.

Plants can synthesize auxin locally, including within leaves, through the highly conserved TAA/YUC pathway^33,34. HAN2 may then transport the auxin synthesized in mature leaves to other tissues. This hypothesis was supported by results from leaf-feeding experiments using d₅-IAA, which showed that the relative transfer rate of d₅-IAA was significantly higher in WT plants than in han2-2 mutants (Fig. 2f, g). Auxin quantification further revealed that auxin content in the young leaves of NIL-HAN2^NPB was significantly lower than that in 93-11(Fig. 2h). and similarly, han2 mutants had significantly lower auxin content in young leaves compared with WT (Fig. 2i). An analysis of whole-plant auxin levels showed that NIL-HAN2^NPB, which has reduced HAN2 expression both before and after chilling treatment, exhibited significantly lower auxin content than 93-11 (Supplementary Fig. 11a). Similarly, han2-2 mutants displayed notably lower auxin levels than WT plants (Supplementary Fig. 11b), whereas overexpression lines showed increased auxin accumulation (Supplementary Fig. 11c). These findings indicate that HAN2/OsABCB5 not only facilitates the transport of auxin from mature leaves to young leaves but also influences overall auxin levels throughout the plant.

HAN2 regulates chilling tolerance potentially via auxin-mediated signalling pathway

Auxin promotes the polyubiquitination and subsequent degradation of Aux/IAA proteins via the 26S proteasome, thereby releasing auxin response factors (ARFs) to activate or repress the expression of downstream responsive genes³⁵. To determine whether HAN2 regulates chilling tolerance through auxin signal transduction, chilling tolerance was assessed in different HAN2 genotypes following exogenous auxin application. In the absence of exogenous auxin, NIL-HAN2^NPB exhibited a significantly higher survival rate than 93-11, and han2 mutants showed significantly greater survival than WT plants. However, in the presence of exogenous auxin, the differences in chilling tolerance between NIL-HAN2^NPB and 93-11 as well as between han2 and WT were reduced (Fig. 3a–d). Previous auxin measurements confirmed that NIL-HAN2^NPB and han2 plants had significantly lower auxin levels compared with 93-11 and WT, respectively (Supplementary Fig. 11a, b). HAN2 was also found to regulate the expression of OsYUC8 (Supplementary Fig. 12a–d), which influences local auxin biosynthesis. Notably, auxin content was significantly lower in osyuc8 mutants than in WT plants, and this reduction was associated with enhanced chilling tolerance (Supplementary Fig. 13a–e). Together, these findings demonstrate that HAN2 negatively regulates chilling tolerance in rice through modulation of auxin signalling.

**Fig. 3: Elucidating *HAN2*-mediated chilling tolerance via auxin signalling.**

To investigate how HAN2 regulates chilling tolerance in rice, the key chilling tolerance gene OsMAP1/OsMPK3^22,23,24,25 was identified among the differentially expressed genes in chilling stress transcriptomes (Supplementary Fig. 14a–e). Compared to their respective controls, 93-11 or WT, OsMAP1/OsMPK3 was significantly upregulated in NIL-HAN2^NPB and han2 mutants, suggesting that HAN2 negatively regulates its expression (Supplementary Fig. 14e). This result was consistent with findings from RT-qPCR analysis (Fig. 3e, f and Supplementary Fig. 15a–c). Conversely, no differences in OsARF24 expression were observed among NIL-HAN2^NPB, han2 mutants, and overexpression lines compared with their respective controls (Supplementary Fig. 15d–f). It has been reported that OsMAP1/OsMPK3 enhanced chilling tolerance in rice by promoting trehalose accumulation²³. We therefor measured trehalose levels under chilling stress and found that the levels were higher in NIL-HAN2^NPB than in 93-11 as well as higher in han2 mutants than in WT (Supplementary Fig. 16a, b). Conversely, trehalose levels were significantly lower in overexpression lines than in WT under chilling stress (Supplementary Fig. 16c). These results indicate that HAN2 negatively regulates the expression of OsMAP1/OsMPK3, which in turn modulates trehalose accumulation and ultimately influences chilling tolerance in rice.

As an auxin transporter, HAN2 may regulate the expression of OsMAP1/OsMPK3 through auxin signal transduction. ARFs are key components of this signalling and can specifically bind to auxin response elements to activate or repress the transcription of downstream genes³⁵. Using a dual-luciferase reporter system in rice protoplasts, OsARF24 was found to inhibit OsMAP1/OsMPK3 expression (Supplementary Fig. 17). Online predictions using New PLACE revealed an ARF-binding element (TGTCTC) in the upstream region of OsMAP1/OsMPK3, which may serve as the binding site for OsARF24. Mutation of this element significantly reduced the inhibitory effect of OsARF24 (Fig. 3g). Subsequent assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation followed by qPCR (ChIP-qPCR), confirmed that OsARF24 directly binds to the ARF element in the OsMAP1/OsMPK3 promoter (Fig. 3h–j and Supplementary Fig. 18). Expression of OsMAP1/OsMPK3 was significantly lower in OsARF24-overexpressing lines (OsARF24-Flag/ZH11) than in WT (Fig. 3k). To assess the functional relationship between OsARF24 and OsMAP1/OsMPK3, we evaluated chilling tolerance in osarf24, OsARF24-Flag/ZH11, osmap1, and osmap1/osarf24 plants, with and without exogenous auxin. Compared with their respective WT controls, OsARF24-Flag/ZH11, osmap1, and osmap1/osarf24 plants displayed increased sensitivity to chilling stress, whereas osarf24 mutants exhibited enhanced tolerance in the absence of auxin (Supplementary Fig. 19a–h and Supplementary Fig. 20a–e). Under auxin treatment, OsARF24-Flag/ZH11, osmap1, and osmap1/osarf24 plants showed further sensitivity, although the differences in chilling response compared with WT were reduced. These results suggest that OsARF24 acts upstream of OsMAP1/OsMPK3. To explore the relationship between auxin and the OsARF24 protein, non-denaturing western blot analysis was performed using total protein extracted from mature leaves of OsARF24-Flag/ZH11 plants. The results showed that auxin-treated plants had higher levels of free OsARF24-Flag protein compared with untreated controls (Fig. 3l), suggesting that elevated auxin levels promote the release of OsARF24, thereby enhancing its regulatory effect on target genes. Furthermore, we generated transgenic lines expressing Flag-tagged OsARF24 in the han2 mutant background (OsARF24-Flag/han2). Expression of OsMAP1/OsMPK3 was significantly lower in OsARF24-Flag/han2 plants than in han2 controls (Supplementary Fig. 21a). Non-denaturing western blot analysis using protein from mature leaves of OsARF24-Flag/ZH11 and OsARF24-Flag/han2 plants revealed that free OsARF24-Flag protein levels were higher in OsARF24-Flag/han2 plants (Supplementary Fig. 21b). Collectively, these findings indicate that HAN2 negatively regulates chilling tolerance in rice potentially through auxin-mediated signalling pathway.

Copia LTR-mediated epigenetic suppression of HAN2 in temperate japonica rice

Within the delimited genomic region, three putative genes were identified (Fig. 1c, Supplementary Fig. 3 and Supplementary Table 1). Among them, LOC_Os01g50090 encodes a Copia LTR located 0.5 kb downstream of the 3′UTR of HAN2, which is absent in the indica cultivar 93-11. Studies have shown that retrotransposons may influence the expression of the neighbouring gene³⁶. To assess its impact on HAN2 expression, we used CRISPR/Cas9 to delete the retrotransposon in the temperate japonica rice cultivar ZH11 (Supplementary Fig. 22a, b). The resulting line, HAN2^-LTR, carried a complete deletion of the retrotransposon (Supplementary Fig. 22c) and exhibited HAN2 expression level approximately twice those of WT (Fig. 4a). HAN2^-LTR plants were also more sensitive to chilling stress, displaying a 50.0% lower survival rate compared with WT plants following chilling treatment (Fig. 4b, c). Additionally, we compared promoter polymorphisms of HAN2 between 93-11 and NPB (Supplementary Fig. 23a). However, no significant difference in HAN2 promoter activity was observed between the two genotypes in rice protoplasts (Supplementary Fig. 23b). These findings indicate that the significant difference in HAN2 expression between indica and temperate japonica rice is attributable to the presence or absence of the Copia LTR downstream of HAN2, rather than to promoter variation.

**Fig. 4: CRISPR/Cas9-based dissection of *HAN2*-downstream *Copia* LTR in gene repression.**

To further validate the effect of the Copia LTR on HAN2 function, we compared HAN2 expression and chilling tolerance across rice varieties with and without the retrotransposon. HAN2 expression was generally higher in five indica rice varieties lacking the LTR than in five temperate japonica rice varieties carrying the LTR (Supplementary Fig. 5b), whereas chilling tolerance exhibited the opposite trend (Supplementary Fig. 1a, b). To minimise the influence of ecotype background, we selected two groups of tropical japonica rice varieties—those with and without the LTR—for further analysis. HAN2 expression was significantly higher in tropical japonica varieties lacking the retrotransposon, and these varieties exhibited weaker chilling tolerance than those carrying the LTR (Fig. 4d, e). Collectively, these results demonstrate that the Copia LTR in japonica rice downregulates HAN2 expression and enhances chilling tolerance.

Insertion of the Copia LTR may influence the DNA methylation status of HAN2. To test this hypothesis, we analysed HAN2 methylation levels using a whole-genome DNA methylation database for the temperate japonica rice cultivar NPB and the indica rice cultivars MH63 and ZS97, both of which lack the retrotransposon (http://glab.hzau.edu.cn/RiceENCODE/). DNA methylation within the gene body of HAN2 was higher in the two indica varieties compared with the japonica variety (Supplementary Fig. 24a). To further investigate this, we performed bisulfite sequencing PCR to assess DNA methylation differences in HAN2 between 93-11 and NIL-HAN2^NPB and between WT and HAN2-LTR (Supplementary Fig. 24b). In the comparison between 93−11 and NIL-HAN2^NPB, CG methylation was generally higher in indica rice, except at the second position (Supplementary Fig. 24c). However, methylation levels between HAN2^-LTR and WT showed no significant differences, except at the fourth position where WT exhibited higher DNA methylation than HAN2^-LTR (Supplementary Fig. 24d). These findings suggest that DNA methylation within the HAN2 gene body differs between the indica and temperate japonica alleles, but this variation is not attributable to the downstream Copia LTR insertion.

The Copia LTR may influence histone methylation at the HAN2 locus. To explore this, we analysed the levels of histone modifications H3K4, H3K27, and H3K36 in the gene body region across different HAN2 genotypes. Active histone marks (H3K4me3 and H3K36me3) were more enriched in the gene body of the HAN2 indica allele than in the temperate japonica allele (Fig. 4f), whereas the repressive mark H3K27me3 was more enriched in the temperate japonica allele (Supplementary Fig. 25a). ChIP-qPCR analysis showed significantly higher enrichment of H3K4me3 and H3K36me3 in the HAN2^-LTR mutant compared with WT, suggesting that the insertion of the Copia LTR reduces the enrichment of active histone modifications (Fig. 4g–i). However, H3K27me3 enrichment was also higher in HAN2^-LTR than in WT (Supplementary Fig. 25b), which contrasts with the comparison between 93-11 and NIL-HAN2^NPB. Collectively, these results indicate that the downstream insertion of the Copia LTR in temperate japonica rice reduces active histone modifications in the HAN2 region, thereby suppressing HAN2 expression.

Copia LTR downstream of HAN2 enhances rice adaptation to temperate habitats

To examine the distribution of the Copia LTR near HAN2, we first genotyped a population comprising various rice ecotypes and wild rice accessions using PCR. The Copia LTR was present in nearly all temperate japonica rice varieties, 50.0% of tropicaljaponica varieties, and 27.5% of wild rice accessions but was absent in indica rice (Supplementary Fig. 26a). To confirm these observations, we analysed data from the 3000 Rice Genomes Project³⁷ and obtained consistent results (Fig. 5a and Supplementary Data 1, 2). These findings suggest that the Copia LTR adjacent to HAN2 in temperate japonica rice likely originated from wild rice ancestors and that HAN2 underwent differentiation between indica and japonica subspecies during domestication.

Fig. 5: Population genetic evidence for adaptive evolution in *HAN2.*

To determine whether the Copia LTR contributed to the adaptation of japonica rice to temperate environments, we first analysed fixation indices (F_ST), which reflect the level of genetic differentiation between populations. Within a 6.0-Mb region (26–32 Mb) surrounding the HAN2 locus, a strong differentiation was observed between indica and japonica rice, with the highest F_ST (Ind, Jap) value reaching 0.71 (Fig. 5b), whereas the genome-wide F_ST (Ind, Jap) value was only 0.55 (Supplementary Fig. 26b). In the region adjacent to HAN2 (26–32 Mb), the F_ST value between temperate japonica (tej) and tropical japonica rice (trj) carrying the retrotransposon (trj⁺) was low (0.10), whereas the F_ST values for tej vs. trj⁻ (0.78) and trj⁺ vs. trj⁻ (0.71) were substantially higher (Fig. 5c and Supplementary Fig. 26c, d). These findings indicate that the retrotransposon may have played a role in the adaptation of japonica rice to temperate climates. This inference was further supported by haplotype analysis of a 1.0 Mb region (28.0–29.0 Mb) surrounding HAN2 using data from the 3000 Rice Genomes Project³⁷ (Fig. 5d). Indica rice lacking the retrotransposon shared a similar haplotype block with tropical japonica rice without the retrotransposon, whereas temperate japonica rice carrying the retrotransposon exhibited a distinct haplotype block, which was also shared by tropical japonica rice with the retrotransposon (Fig. 5d and Supplementary Fig. 26e). Nucleotide diversity analysis revealed that temperate japonica rice has lower genetic diversity in the HAN2 region compared with wild and indica rice (Fig. 5e). The Tajima’s D value for the HAN2 region in temperate japonica rice was −2.65 (Supplementary Fig. 26f), exceeding the 5% significance threshold, indicating a marked reduction in genetic diversity—likely due to positive selection or population migration. Additionally, among tropical japonica rice, varieties with the retrotransposon exhibited lower diversity in the HAN2 region than those without it (Fig. 5f). These results suggest that the genomic region containing the Copia LTR downstream of HAN2 underwent selection during the domestication of temperate japonica rice, contributing to its adaptation to temperate environments.

We conducted a geographical distribution analysis of rice varieties across Asia. The results showed that the prevalence of the Copia LTR varied by region, ranging from 3.6% in Sri Lanka at low latitudes to 100% in Korea at high latitudes, with intermediate frequencies of 30.4%, 12.6%, and 14.7% in China, India, and Laos, respectively (Fig. 5g). Among the 1039 indica rice varieties analysed, only 19 carried the retrotransposon. In contrast, 282 out of 285 temperate japonica rice varieties harboured the Copia LTR (Fig. 5a). Indica rice is predominantly cultivated in low-latitude, low-altitude regions, whereas temperate japonica rice is grown in high-latitude, high-altitude environments. These findings suggest that the Copia LTR downstream of HAN2 plays a crucial role in the adaptation of temperate japonica rice to colder climates, indicating that HAN2 was likely subject to selection pressure from chilling stress during domestication.

Suppression of HAN2 confers dual-stage chilling tolerance and tillering advantage in rice

In addition to the seedling stage, we evaluated the impact of HAN2 on chilling tolerance during the booting stage. Under normal growth conditions, no significant differences in seed setting rates were observed between 93-11 and NIL-HAN2^NPB or between WT and han2. However, following chilling treatment at the booting stage, the seed setting rate of NIL-HAN2^NPB was significantly higher than that of 93-11, and han2 mutants exhibited higher seed setting rates than WT (Fig. 6a–f). Given that HAN2 negatively regulates seedling chilling tolerance in rice, we speculated that HAN2 may also regulate chilling tolerance during the booting stage via the same mechanism. Then, we measured auxin content in young spikelets of NIL-HAN2^NPB and han2 mutants under chilling treatment. Compared with their respective controls, auxin levels were significantly lower in both NIL-HAN2^NPB and han2 mutants (Fig. 6g, j). RT-qPCR analysis showed that OsMAP1/OsMPK3 expression was higher in young spikelets of NIL-HAN2^NPB and han2 under chilling stress compared with the controls (Fig. 6h, k), whereas OsARF24 expression did not differ significantly between NIL-HAN2^NPB, han2, and their corresponding controls (Supplementary Fig. 27a, b). Additionally, trehalose content in the young spikelets of NIL-HAN2^NPB and han2 under chilling stress showed a similar trend to that observed during seedling chilling tolerance (Fig. 6i, l). Together, these results indicate that HAN2 negatively regulates chilling tolerance in rice at the booting stage, also potentially through auxin-mediated signalling pathway. Furthermore, transgenic lines overexpressing HAN2 under the control of either the 35S promoter or the native HAN2 promoter, as well as CRISPR/Cas9-edited HAN2^-LTR lines, exhibited significantly lower seed setting rates under chilling stress at the booting stage compared with their respective controls (Supplementary Figs. 28a–f and Supplementary Figs. 29a–c). These findings demonstrate that HAN2 negatively affects chilling tolerance in rice at both the seedling and booting stages.

**Fig. 6: Functional analysis of *HAN2* regulation on chilling tolerance at rice booting stage.**

Auxin plays a key role in regulating plant branching or tillering³⁸. We hypothesised that HAN2 influences rice tillering and, consequently, yield. Field assays conducted in Changsha during the summer of 2023 showed that NIL-HAN2^NPB, which has reduced HAN2 expression, produced 1.3 more tillers than 93-11, resulting in a 16.3% increase in yield (Supplementary Fig. 30a–e). Similarly, han2 mutants produced 3.8 more tillers than WT plants, leading to a 15.2% increase in yield (Supplementary Fig. 30f–j). Field trials conducted in Hainan during the winter of 2023 further confirmed these results: yield per plant in NIL-HAN2^NPB was 14.7% higher than that in 93-11 (Supplementary Fig. 31a–f), and yield per plant in han2 was 28.2% higher than that in WT (Supplementary Fig. 32a–f). These findings highlight the potential of the temperate japonica rice allele or a loss-of-function HAN2 allele to not only enhance chilling tolerance at both the seedling and booting stages but also improve rice yield through increased tillering, offering substantial value for breeding programmes aimed at enhancing and stabilising rice production.

Discussion

Rice cultivated in Asia primarily consists of two subspecies: indica and japonica rice. Typically, temperate japonica rice varieties exhibit greater adaptability to chilling stress than indica varieties^1,4. However, the molecular basis underlying this adaptability remains incompletely understood, and the role of auxin in mediating the chilling tolerance of temperate japonica rice remains to be fully clarified. Herein, we isolated a chilling tolerance gene, HAN2, from temperate japonica rice using a map-based cloning approach. HAN2 encodes an ABCB transporter, OsABCB5, which functions in auxin efflux. The temperate japonica allele of HAN2 contains a downstream retrotransposon insertion that leads to reduced methylation of lysine residues at positions 4 and 36 on histone H3 in the HAN2 region. This epigenetic modification results in lower transcriptional activity and subsequently alters auxin transport. Compared with the indica allele, the temperate japonica HAN2 allele confers enhanced chilling tolerance at both the seedling and booting stages, largely due to the presence of the retrotransposon insertion (Fig. 7). Our findings further reveal that HAN2/OsABCB5 negatively regulates rice chilling tolerance potentially through an auxin-cascade involving OsARF24 andOsMAP1/OsMPK3. However, since we don’t have direct genetic evidence (i.e. higher order mutants data), we cannot state that HAN2 could form any signalling module with its potential downstream interactor(s) to mediate chilling adaptation.

**Fig. 7: Possible working model of *HAN2*-mediated chilling adaptation in rice.**

Interestingly, HAN2 regulates auxin levels in plants, similar to other members of the ABCB transporter family, such as SD8³⁹. However, the mechanism by which HAN2 influences auxin content remains unclear. The tryptophan-dependent pathway is the primary route for auxin biosynthesis, with YUCCA-encoded flavin monooxygenases catalysing the final rate-limiting step³³. To explore this further, we analysed the expression patterns of YUCCA family members. Among them, OsYUC8 exhibited an expression pattern in leaves similar to that of HAN2 (Supplementary Fig. 12a) and has been reported to influence leaf development (Supplementary Data 3)⁴⁰, suggesting that OsYUC8 is the key target modulated by HAN2 in leaf tissues. RT-qPCR analysis confirmed that OsYUC8 expression was significantly lower in NIL-HAN2^NPB than in 93-11 (Supplementary Fig. 12b), significantly lower in han2 mutants than in WT plants (Supplementary Fig. 12c), and significantly higher in HAN2-overexpressing lines than in WT (Supplementary Fig. 12d). Detached leaf incubation experiments using d₅-Trp further revealed that d₅-IAA synthesis was significantly reduced in osyuc8 mutants compared with WT (Supplementary Fig. 13b). Moreover, osyuc8 plants exhibited lower auxin content and a higher survival rate following chilling treatment than WT plants (Supplementary Fig. 13c-e). Based on these results, we propose that OsYUC8 plays a regulatory role in auxin biosynthesis within mature leaves and that HAN2 promotes OsYUC8 expression via an as-yet unidentified feedback mechanism, thereby enhancing localised auxin synthesis.

The d₅-IAA leaf-feeding experiment demonstrated that the relative transfer rate of d₅-IAA was significantly higher in WT plants than in han2 mutants across multiple leaves (Fig. 2f-g). This finding indicates that HAN2 facilitates the movement of d₅-IAA from mature source leaves to other plant tissues. Although auxin is primarily synthesised in young tissues, our results show that mature leaves can also produce auxin (Supplementary Fig. 13b). The high expression of HAN2 in mature leaves likely plays a critical role in transporting auxin synthesised in these tissues to other parts of the plant. This inference is further supported by auxin level measurements in the third to fifth leaves of NIL-HAN2^NPB and han2 under both normal and chilling conditions (Supplementary Fig. 33a-d). The main auxin transport proteins located on the plasma membrane include members of the AUX1/LAX, ABCB/PGP, and PIN families, which coordinate the intracellular and extracellular transport of auxin⁴¹. Auxin uptake is primarily mediated by the efficient IAA⁻/H⁺ symport mechanism, facilitated by members of the AUX1/Like-AUX1 family⁴². Transcriptomic analysis of NIL-HAN2^NPB and han2 revealed no significant differences in the expression levels of OsAUX genes across different HAN2 genotypes, suggesting that HAN2 does not influence these transporters (Supplementary Fig. 34a-b). Additionally, recent studies have shown that ABCB transporters can promote auxin efflux independently of PIN proteins⁴³. However, the efflux of auxin mediated by PINs typically depends on a synergistic mechanism requiring co-localisation with ABCB transporters⁴³.

The insertion of Copia LTR results in reduced enrichment of the active histone marks H3K4me3 and H3K36me3, which collectively downregulate HAN2 expression and enhance chilling tolerance in both NPB and the HAN2^-LTR mutant (Fig. 4f, Supplementary Fig. 1a-b, and Fig. 4b, c). To date, no studies have specifically reported the effects of Copia retrotransposon insertions on histone methylation. However, recent research has shown that a solo-LTR insertion can increase DNA methylation and promote the accumulation of the repressive histone mark H3K9me2, which together suppress GY3 expression and lead to higher grain yield in the 02428 cultivar and other genetic backgrounds⁴⁴. It is well established that transposable elements can influence the epigenetic regulation of nearby coding genes by promoting the spread of histone methylation marks such as H3K9me2⁴⁵. Therefore, the insertion of the Copia LTR downstream of HAN2 in temperate japonica rice may similarly affect histone methylation through a diffusion-like mechanism, thereby modulating HAN2 expression.

With the intensification of global climate change, achieving high and stable crop yields is essential for ensuring food security. To this end, identifying key genes that contribute to desirable agronomic traits in grain crops is critical. Although numerous genes associated with high yield or abiotic stress tolerance have been identified⁴⁶, genes that simultaneously confer high and stable yield remain limited. The temperate japonica rice allele HAN2/OsABCB5 represents a promising example, conferring enhanced chilling tolerance at both the seedling and booting stages, along with increased tiller number. Following chilling treatment, OsMAP1/OsMPK3 expression in NIL-HAN2^NPB was consistently higher than that in 93-11 at both stages (Fig. 3e, Supplementary Fig. 15a, and Fig. 6h), and trehalose content was significantly greater in NIL-HAN2^NPB than in 93-11 (Supplementary Fig. 16a and Fig. 6i). These findings suggest that HAN2 enhances chilling tolerance across developmental stages, potentially through auxin-mediated signalling pathway. Interestingly, no significant difference in tiller number was observed between OsARF24 overexpression lines and WT (Supplementary Fig. 35), and yield-related traits such as plant height and effective tiller number remained unchanged in OsMAP1 knockdown and overexpression lines compared with WT⁴⁷. These results indicate that the regulatory networks by which HAN2 controls chilling tolerance and tiller number are distinct. The advantageous HAN2 allele originated from the insertion of an Copia LTR downstream of the gene, an element traceable to wild rice. This LTR is present in 5.5% of wild rice, 50.0% of tropical japonica, and 97.3% of temperate japonica rice varieties (Supplementary Fig. 26a). Among indica rice, the insertion is nearly absent, while only 14.3% of the Aus ecotype—believed to have undergone hybridisation with japonica rice during domestication⁴⁴—carry the insertion. It is therefore hypothesised that the LTR insertion in HAN2 originated from wild rice and was selected for in temperate japonica rice to support adaptation to cooler climates. Introgression of the temperate japonica HAN2 allele into indica rice improves tillering, grain yield, and chilling tolerance at both the seedling and booting stages, thereby contributing to high and stable yield performance. In japonica rice, gene editing techniques can be used to knock out HAN2, resulting in improved tillering, yield, and chilling tolerance. Thus, HAN2 represents a valuable genetic resource for achieving high and stable rice production under climate variability.

Methods

Plant materials and phenotypic evaluations

A set of chromosome segment substitution lines was developed using the temperate japonica rice cultivar NPB as the donor and the indica cultivar 93-11 as the recipient. From this population, a near-isogenic line, NIL-HAN2^NPB, was selected and subsequently backcrossed to 93-11 and self-fertilised to generate a fine-mapping population for HAN2.

To evaluate chilling tolerance at the seedling stage, 7 day-old seedlings of indica or japonica rice were exposed to 10 °C or 4 °C in a light incubator for 4–6 days. Following this treatment, seedlings were transferred to a growth chamber set at 28 °C/25 °C with a 13 h/11 h light/dark cycle for a 3–7 day recovery period, after which survival rates were recorded. Plant mortality was defined as the inability to produce new foliage during the recovery phase under ambient conditions after chilling exposure, ultimately resulting in complete desiccation.

To evaluate chilling tolerance at the booting stage, plants at the booting phase—with a distance of −3 to 0 cm between the lamina joints of the flag leaf and the second leaf—were subjected to chilling treatment in a walk-in climate chamber (19 °C for indica and 16 °C for japonica, 12 h light/12 h dark) for 7 days. Following treatment, the plants were returned to normal growth conditions (28 °C/25 °C, 13 h light/11 h dark) and maintained until full maturity. Seed setting rates were determined using a minimum of four biological replicates per genotype.

Fine mapping of the HAN2 locus

To fine-map HAN2, a genetic segregation population (BC₁F₂) was generated by backcrossing NIL-HAN2^NPB with 93-11, followed by self-fertilisation. Approximately 3200 individuals from this population were genotyped using seven pairs of molecular markers. By correlating the genotypes of markers M4 and M5 with the chilling tolerance phenotypes of six key recombinant lines (R1–R6), HAN2 was mapped to a 30.0-kb genomic interval.

Vector construction and genetic transformation

For the genetic complementation of HAN2, the HAN2 coding sequence along with its 2.0 kb native promoter was amplified from the 93-11 genome using primers CP-HAN2-F and CP-HAN2-R. The resulting fragment was inserted into the pCAMBIA1301 vector to generate a fusion construct in which HAN2 is driven by its native promoter.

To construct an overexpression vector, the full-length HAN2 gene, including all introns, was amplified from the NPB genome using primers HAN2-OE-F and HAN2-OE-R. The amplified fragment was then cloned into the pCAMBIA1300-EGFP expression vector under the control of the constitutive 35S promoter.

For the HAN2 knockout, a CRISPR/Cas9-based vector was designed with an sgRNA targeting the exon of HAN2 under the OsU6a promoter.

To knock out the Copia LTR, a CRISPR/Cas9-based vector containing two sgRNAs was constructed: one sgRNA targeting the left flanking sequence of the retrotransposon was driven by the OsU6a promoter, and the other targeting the right flanking sequence was driven by the OsU6b promoter. The complementation vector was introduced into the NIL-HAN2^NPB line via Agrobacterium-mediated transformation, whereas the remaining constructs were transformed into the temperate japonica rice cultivar ZH11.

RNA extraction and expression analysis

Total RNA was extracted using the AG RNAex Pro RNA reagent (Accurate, China, AG21102). The extracted RNA was reverse transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme). RT-qPCR was then performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). Gene expression levels were analysed using the ∆∆Ct method based on 3–4 biological replicates. Statistical significance was determined using Student’s t-test.

RNA-seq analysis

Rice shoots were harvested and immediately frozen in liquid nitrogen for RNA-seq analysis. Three biological replicates were included for each sample. RNA sequencing was conducted by BGI using the MGI platform. For data analysis, processed reads were aligned to the reference genome MSURelease7.0⁴⁸ using Hisat2 v2.0.1⁴⁹. Gene quantification was performed with HTSeq v0.6.1⁵⁰, and differential expression analysis was performed using DESeq2⁵¹. Differentially expressed genes were identified based on a P-value < 0.05 and an absolute log₂ fold change ( | log₂FC | ) ≥ 1. Gene Ontology enrichment analysis was conducted using the DAVID database (https://david.ncifcrf.gov/home).

Subcellular localisation

The constructed vectors 35S::HAN2-GFP and 35S::GFP were introduced into rice protoplasts via PEG-mediated transformation. Following transformation, the protoplasts were stained with 100 μM FM4-64 (a plasma membrane marker) for 1 min, washed with W5 solution, and examined for GFP fluorescence using a confocal laser scanning microscope (LSM880, Carl Zeiss).

DNA methylation analysis

Genomic DNA was extracted from the third leaf of 2 week-old rice seedlings using the SDS method. CpG island regions were predicted and designed using MethPrimer, and bisulfite sequencing PCR (BSP) primers for HAN2 were designed accordingly (Supplementary Data 4; http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). The extracted DNA was treated with sodium bisulfite using the EpiTect Bisulfite Conversion Kit (Qiagen). PCR products amplified with BSP primers were ligated into the pEASY Blunt cloning vector (TransGen Biotech), and randomly selected clones were sequenced. CG methylation levels were analysed using Sequencher 5.4.5 software.

ChIP assay

Using ChIP-seq datasets of 93-11 and NPB (SRR10751611, SRR10751742, SRR10751615, SRR10751746, SRR094791, SRR1827950) available in the NCBI public repository^52,53,54, we analysed histone modifications H3K4me3, H3K27me3, and H3K36me3 in the HAN2 region.

Leaf samples (4 g) were collected from 2 week-old rice seedlings and cross-linked with 1% formaldehyde. The cross-linking reaction was quenched with 0.125 mol/L glycine. ChIP assays were performed using the EpiQuik Plant ChIP Kit (Epigentek, Brooklyn, NY, USA) with the following antibodies: anti-Flag (#14793, Cell Signalling Technology, Danvers, MA, USA; dilution 1:50), anti-H3K4me3 (A22146, ABclonal, Wuhan, China; dilution 1:50), anti-H3K27me3 (A16199, ABclonal, Wuhan, China; dilution 1:50), and anti-H3K36me3 (A20379, ABclonal, Wuhan, China; dilution 1:50). Chromatin was sheared to fragments of <1000 bp via sonication. Enriched DNA was analysed via RT-qPCR using primers targeting the ARF-motif site in the OsMAP1/OsMPK3 promoter or primers spanning the HAN2 exon region. ChIP-qPCR assays for H3K4me3, H3K27me3, and H3K36me3 were conducted to compare lines such as WT and HAN2^-LTR. To confirm OsARF24 binding to the OsMAP1/OsMPK3 promoter, ChIP-qPCR was performed in the p35S::ARF24-Flag overexpression line. Chromatin precipitated with normal mouse IgG (provided in the kit) was used as a negative control. All primers used are listed in Supplementary Data 4.

Luciferase transient transcriptional activity assay

Using NPB and 93-11 as templates, a 2.0 kb promoter region upstream of the HAN2 start codon was amplified and cloned into the pGreenII0800-LUC vector to construct the reporter plasmid. The 35S promoter in the pGreenII0800-LUC vector was then replaced with the HAN2 promoter to generate the pHAN2::LUC construct. The reporter plasmids were introduced into rice protoplasts and incubated in the dark at 28 °C for 12 h. Protoplasts were then harvested and lysed using the lysis buffer provided in the Dual-Luciferase Reporter Assay Kit (Vazyme, DL101-01). Firefly luciferase activity was measured according to the manufacturer’s instructions. Renilla luciferase, driven by the 35S promoter in the pGreenII0800-LUC vector, served as an internal control. Relative firefly luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase activity for each sample.

Determination of auxin content

The method for extracting auxin was carried out according to previously described with minor modifications⁵⁵.To analyze the indole-3-acetic acid (IAA) content in 2-week-old rice seedlings under both normal and chilling treatment conditions (4 °C or 10 °C), samples of the shoots or leaves were collected and immediately frozen in liquid nitrogen. 0.1 g sample was weighed into a 1.5 mL tube, mixed with 750 μL cold extraction buffer (methanol: ddH₂O:acetic acid, 80:19:1, v/v/v), vigorously shaken on a shaking bed for 16 h at 4 °C in dark, and then centrifuged at 14,000 × g for 15 min at 4 °C. The supernatant was carefully transferred to a new 1.5 mL tube and the pellet was remixed with 400 μL extraction buffer, shaken for 4 h at 4 °C, and centrifuged. The two supernatants were combined and filtered the mixture into a sample bottle using a nylon filter membrane with a diameter of 13 millimetres and a pore size of 0.22 micrometres (Nylon 66; Tianjin Jinteng Experimental Equipment Co., Ltd.). The samples in the sample bottle will be analyzed for auxin determination using AB SCIEX QTRAP 5500 mass spectrometer.

Determination of trehalose content

The trehalose content was determined according to previously described with minor modifications²³. Seedlings (0.5 g) were collected at the seedling stage and subjected to low-temperature treatment—either before or after exposure—for 24 h at 4 °C for japonica rice or 10 °C for indica rice. At the booting stage, young panicles (0.5 g) were collected and treated at 19 °C for 24 h in indica rice, either before or after chilling treatment. Samples were ground in liquid nitrogen, and 2 mL of 80% (v/v) ethanol was added. Following centrifugation at 13,000 × g for 15 min, the supernatant was collected and heated in boiling water for 20 min and then re-centrifuged at 13,000 × g for 10 min. The resulting supernatant was dried at 80 °C, and the residue was dissolved in 4 mL of deionised water and stored at −20 °C until analysis. Before high-performance liquid chromatography, the samples were filtered thrice through a 0.22 µm sterile filter. Each experiment included 3–4 biological replicates, and statistical significance was determined using Student’s t-test.

Yeast transport experiment

The control vector pYES-MOB-GFP-IST2 and the construct pYES-MOB-HAN2-GFP-IST2 were introduced into the yeast strain INSCV1. For the d₅-IAA uptake assay, individual yeast colonies were cultured in SD-Ura liquid medium containing 2% glucose at 30 °C with shaking at 200 rpm until the optical density (OD₆₀₀) reached 0.8. Cells were then harvested through centrifugation at 4 °C and 3500 × g for 10 min, washed thrice with sterile water, and resuspended in SD-Ura medium containing 2% lactose, adjusting the OD₆₀₀ to 20. d₅-IAA was then added to a final concentration of 150 μM, and the cell suspension was incubated at 30 °C with shaking at 200 rpm for 3 h. After incubation, cells were washed with 10% methanol followed by sterile water, dried, and subjected to isotope ratio mass spectrometry (MAT253, Thermo Fisher Scientific). Three independent biological replicates were performed for each sample.

In the d₅-IAA efflux assay, yeast clones were cultured in SD-Ura liquid medium containing 2% glucose at 30 °C with shaking at 200 rpm until an OD₆₀₀ of 0.8 was reached. The subsequent steps followed the same protocol as the uptake experiment, except that a portion of the harvested cells was incubated for 12 h in SD-Ura medium containing 2% lactose before further processing. All remaining steps were identical to those used in the uptake assay, including the use of three biological replicates for each sample.

Transport activity assay in Xenopus oocytes

Using overlapping PCR, we constructed pT7::GFP and pT7::GFP-HAN2 expression modules for injection into Xenopus oocytes. cRNA was synthesised by in vitro transcription according to the manufacturer’s protocol (Yeasen, 10623ES50). A volume of 50 nL of GFP, GFP-HAN2 cRNA, or water (negative control) was injected into selected oocytes using a Nanoject II automatic injector. The oocytes were incubated in modified barth’s solution (MBS) at 18 °C for 24 h before transport activity assays. For the uptake experiment, five injected Xenopus oocytes were transferred to MBS solution containing 150 μM d₅-IAA and incubated at 18 °C for 4 h. After incubation, oocytes were washed thrice with fresh MBS solution, dried, and prepared for further analysis. Three biological replicates were conducted. For the efflux experiment, injected oocytes were first incubated in MBS solution containing 150 μM d₅-IAA at 18 °C for 12 h. Afterwards, they were washed thrice with fresh MBS solution and transferred to new MBS solution. Five oocytes were collected at 0 and 4 h post-transfer, washed thrice, and dried. Both uptake and efflux samples were analysed using an isotope ratio mass spectrometer (MAT253, Thermo Fisher Scientific).

Protoplast transformation experiment in rice

Protoplasts were isolated from WT and han2-2 plants⁵⁶. The protoplasts were suspended in pre-cooled W5 solution. For the loading experiment, the protoplasts were centrifuged at 4 °C and 100 × g for 10 min. The supernatant was discarded, and the protoplasts were resuspended in pre-cooled W5 solution. Cell concentration was determined using a hemocytometer. Protoplasts were then transferred into 2 mL round-bottom centrifuge tubes, with each tube containing a final d₅-IAA concentration of 75 μM. Twelve tubes were prepared and incubated at 28 °C with gentle shaking at 48 rpm for 0, 0.5, 1, and 2 h. At each time point, three tubes were selected. The protoplasts were centrifuged at 4 °C and 100 × g for 5 min, the supernatant was removed, and the protoplasts were washed 2–3 times with 1 mL of pre-cooled W5 solution and then collected and dried for further analysis.

For the efflux experiment, protoplasts that had been incubated with d₅-IAA for 2 h were washed 2–3 times with pre-cooled W5 solution, resuspended in fresh W5 solution, and transferred to 2 mL round-bottom centrifuge tubes, with 1 mL of protoplast suspension per tube. Twelve tubes were prepared and incubated at 28 °C for 0, 1, 2, and 3 h. At each time point, three tubes were selected. The protoplasts were centrifuged at 4 °C and 100 × g for 5 min, and the supernatant was transferred to a new centrifuge tube for further analysis. The protoplasts were then washed 2–3 times with 1 mL of pre-cooled W5 solution, collected, and dried. Both the loading and efflux samples were analysed using an isotope ratio mass spectrometer.

Feeding of d₅-IAA to rice leaves

ZH11 and han2 seedlings with uniform growth were selected after 3 weeks of cultivation in hydroponic nutrient solution. A 60 μM d₅-IAA solution was applied to the third leaf, positioned approximately 6 cm from the tip, and allowed to absorb for 48 h. After treatment, samples of the treated leaf, leaf sheath, and root tissues were collected, ground into a fine powder, and dried. d₅-IAA levels were measured using isotope ratio mass spectrometry.

GUS histochemical staining and fluorescence immunocytochemistry

The pHAN2::HAN2-GUS fusion expression vector, driven by the HAN2 promoter, was constructed and introduced into NIL-HAN2^NPB rice via Agrobacterium-mediated transformation. Positive transgenic lines were selected for GUS staining and immunofluorescence analysis. GUS histochemical staining was performed on T₃ homozygous transgenic plants. Leaves and leaf sheaths from seedlings were stained, observed, and photographed using a stereomicroscope. For immunofluorescence analysis, the second leaf and second leaf sheath from 3 week-old seedlings were examined using a GUS antibody (Abcam, AB50148; dilution 1:100). Fluorescent signals were visualised with a confocal laser scanning microscope (LSM880, Carl Zeiss).

Identification of the HAN2-neighbouring Copia LTR

Two pairs of primers were designed to detect the downstream insertion of the Copia LTR in 134 rice varieties, including 28 indica, 14 tropical japonica, 37 temperate japonica, and 55 wild rice accessions. The primer pair F/1 R successfully amplified a 368 bp PCR product from NPB genomic DNA, which contains the retrotransposon, whereas no amplification was observed from 93-11 genomic DNA, as the reverse primer (1 R) specifically binds within the retrotransposon, which is absent in 93-11. Conversely, the primer pair F/2 R amplified a 465 bp PCR product from 93-11 genomic DNA lacking the retrotransposon, whereas no amplification was detected from NPB genomic DNA. This is likely because the target region in NPB spans approximately 3.8 kb, exceeding the amplification range of the PCR conditions used.

Y1H assay

The ARF cis-regulatory element from the OsMAP1/OsMPK3 promoter and its corresponding mutant version (ARFm) were synthesised with three tandem copies and cloned into the pHis2.0 vector via homologous recombination to generate the pHis2–3 × ARF and pHis2–3 × ARFm constructs, respectively. The full-length coding sequence of OsARF24 was amplified using PCR from a rice cDNA library and inserted into the pGADT7 vector via homologous recombination to produce the AD-OsARF24 construct. The Y1H assay was conducted using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Clontech). Briefly, transformants were first plated on SD/-Trp-Leu medium for growth assessment, followed by spotting on SD/-Trp-Leu-His and SD/-Trp-Leu-His media supplemented with 3-amino-1,2,4-triazole for interaction analysis.

EMSA

The coding region of OsARF24 was amplified from a rice cDNA library and cloned into the pGEX-6P-1 vector via homologous recombination. Protein expression and purification were performed according to the manufacturer’s instructions. A biotin-labelled oligonucleotide corresponding to the OsARF24 binding site was synthesised by annealing equimolar complementary oligonucleotides in Taq DNA polymerase buffer. EMSAs were conducted at room temperature using the EMSA kit (20148, Thermo Fisher Scientific). Briefly, biotin-labelled probes were incubated with either GST or GST-OsARF24 in binding buffer for 20 min. For competition assays, 50× and 200× molar excesses of unlabelled competitor probes were included. DNA–protein complexes were separated on 5% native polyacrylamide gels, and biotin signals were detected using the ChemiDoc XRS System (Bio-Rad, USA).

Protein extraction and western blot analysis

Total protein was extracted from the shoots or mature leaves of 15 day-old OsARF24-Flag/ZH11 and OsARF24-Flag/han2 seedlings using a total protein extraction kit (BestBio, BB-3183) according to the manufacturer’s instructions. The extracted proteins were then subjected to non-denaturing western blot analysis. Total OsARF24-Flag protein levels, assessed via SDS-PAGE western blotting, were used as an internal reference. Immunoblot analysis was performed using anti-Flag monoclonal antibodies (CST, D6W5B, dilution 1:1000). All experiments were repeated at least thrice, yielding similar results.

Population genetic analysis of HAN2

To obtain the SNPs required for downstream analyses, SNP calling was performed using the VariantFiltration tool in GATK (v4.2.2.0) with default parameters. F_ST values were calculated using VCFtools (v0.1.16) with a sliding window of 100 kb and a step size of 50 kb. The map shown in Fig. 6g was generated using the ggmap package in R⁵⁷. Nucleotide diversity within the HAN2 region across different populations was calculated using DnaSP v5.10⁵⁸. SNPs were imputed using Beagle (v5.1)⁵⁹, and haplotype blocks were extracted using plotHaps in vcflib (v1.0.1)⁶⁰ and visualised with R (v4.0.2).

Phylogenetic analysis

The full-length protein sequences of ABCB transporters from both rice and Arabidopsis genomes were obtained from the Ensembl Plants database (http://plants.ensembl.org/index.html). Sequence alignment was performed using ClustalW in MEGA7⁶¹. A neighbour-joining phylogenetic tree was then constructed based on the aligned sequences using the Poisson correction method with pairwise deletion of gaps. The reliability of the tree was assessed through bootstrap analysis with 1000 replicates.

Primers

All primers used in this study are shown in the Supplementary Data 4.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10. Data are presented as the mean ± standard error of the mean. For comparisons between two groups, a two-tailed independent samples t-test was used, whereas one-way ANOVA followed by Tukey’s multiple comparisons test was applied for comparisons involving three or more groups.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data supporting the findings of this work are available within this paper and its Supplementary Information files. Genetic materials used in this work are available from the corresponding authors upon request. RNA-Seq and DNA re-sequencing data generated in this study have been deposited in the NCBI/Sequence Read Archive (SRA) database under accession code PRJNA1244014 and PRJNA1244057. ChIP-Seq datasets of 93-11 and NPB are available in the NCBI/Sequence Read Archive (SRA) database under accession code SRR10751611, SRR10751742, SRR10751615, SRR10751746, SRR094791 and SRR1827950. Gene sequence and annotation information is available from the Rice Genome Annotation Project (https://rice.uga.edu/). Source data are provided with this paper.

References

Mao, D. et al. Natural variation in the HAN1 gene confers chilling tolerance in rice and allowed adaptation to a temperate climate. Proc. Natl Acad. Sci. USA 116, 3494–3501 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Li, J. et al. Chilling tolerance in rice: past and present. J. Plant Physiol. 268, 153576 (2022).
Article CAS PubMed Google Scholar
Lu, G. et al. Rice LTG1 is involved in adaptive growth and fitness under low ambient temperature. Plant J. 78, 468–480 (2014).
Article CAS PubMed Google Scholar
Ma, Y. et al. COLD1 confers chilling tolerance in rice. Cell 160, 1209–1221 (2015).
Article CAS PubMed Google Scholar
Zhao, J. et al. A novel functional gene associated with cold tolerance at the seedling stage in rice. Plant Biotechnol. J. 15, 1141–1148 (2017).
Article CAS PubMed PubMed Central Google Scholar
Chen, X. et al. A missense mutation in Large Grain Size 1 increases grain size and enhances cold tolerance in rice. J. Exp. Bot. 70, 3851–3866 (2019).
Article CAS PubMed PubMed Central Google Scholar
Liu, H. et al. OsWRKY115 on qCT7 links to cold tolerance in rice. Theor. Appl. Genet. 135, 2353–2367 (2022).
Article CAS PubMed Google Scholar
Li, Z. et al. Natural variation of codon repeats in COLD11 endows rice with chilling resilience. Sci. Adv. 9, eabq5506 (2023).
Article ADS PubMed PubMed Central Google Scholar
Xia, C. et al. The COG1-OsSERL2 complex senses cold to trigger signaling network for chilling tolerance in japonica rice. Nat. Commun. 14, 3104 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Feng, J. et al. COG2 negatively regulates chilling tolerance through cell wall components altered in rice. Theor. Appl. Genet. 136, 19 (2023).
Article PubMed Google Scholar
Liu, D. et al. COG3 confers the chilling tolerance to mediate OsFtsH2-D1 module in rice. N. Phytol. 241, 2143–2157 (2024).
Article CAS Google Scholar
Sun, S. et al. Domestication-selected COG4-OsbZIP23 module regulates chilling tolerance in rice. Cell Rep. 43, 114965 (2024).
Article CAS PubMed Google Scholar
Luo, W. et al. COLD6-OSM1 module senses chilling for cold tolerance via 2’,3’-cAMP signaling in rice. Mol. Cell 84, 4224–4238.e9 (2024).
Article CAS PubMed Google Scholar
Saito, K. et al. Physical mapping and putative candidate gene identification of a quantitative trait locus Ctb1 for cold tolerance at the booting stage of rice. Theor. Appl. Genet. 109, 515–522 (2004).
Article CAS PubMed Google Scholar
Zhang, Z. et al. Natural variation in CTB4a enhances rice adaptation to cold habitats. Nat. Commun. 8, 14788 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Liu, C. et al. Early selection of bZIP73 facilitated adaptation of japonica rice to cold climates. Nat. Commun. 9, 3302 (2018).
Article ADS PubMed PubMed Central Google Scholar
Li, J. et al. Natural variation of indels in the CTB3 promoter confers cold tolerance in japonica rice. Nat. Commun. 16, 1613 (2025).
Article CAS PubMed PubMed Central Google Scholar
Guo, H. et al. Natural variation of CTB5 confers cold adaptation in plateau japonica rice. Nat. Commun. 16, 1032 (2025).
Article CAS PubMed PubMed Central Google Scholar
Gao, S. et al. CTB6 confers cold tolerance at the booting stage by maintaining tapetum development in rice. Adv. Sci. 12, e2411357 (2025).
Fujino, K. et al. Molecular identification of a major quantitative trait locus, qLTG3-1, controlling low-temperature germinability in rice. Proc. Natl. Acad. Sci. USA. 105, 12623–12628 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, C. et al. A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination. Nat. Commun. 15, 2211 (2024).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, Z. et al. OsMAPK3 phosphorylates OsbHLH002/OsICE1 and inhibits its ubiquitination to activate OsTPP1 and enhances rice chilling tolerance. Dev. Cell 43, 731–743 (2017).
Article CAS PubMed Google Scholar
Lou, Q. et al. Cold‐adaptive evolution at the reproductive stage in Geng/japonica subspecies reveals the role of OsMAPK3 and OsLEA9. Plant J. 111, 1032–1051 (2022).
Article CAS PubMed Google Scholar
Xia, C. et al. Phosphatase OsPP2C27 directly dephosphorylates OsMAPK3 and OsbHLH002 to negatively regulate cold tolerance in rice. Plant Cell Environ. 44, 491–505 (2021).
Article CAS PubMed Google Scholar
Xie, G. et al. Biochemical identification of the OsMKK6-OsMPK3 signaling pathway for chilling stress tolerance in rice. Biochem. J. 443, 95–102 (2012).
Article CAS PubMed Google Scholar
Chen, L. et al. OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice. N. Phytol. 218, 219–231 (2018).
Article CAS Google Scholar
Su, C. et al. A novel MYBS3-dependent pathway confers cold tolerance in rice. Plant Physiol. 153, 145–158 (2010).
Article CAS PubMed PubMed Central Google Scholar
Li, Z. et al. OsGRF6 interacts with SLR1 to regulate OsGA2ox1 expression for coordinating chilling tolerance and growth in rice. J. Plant Physiol. 260, 153406 (2021).
Article CAS PubMed Google Scholar
Zhang, M. et al. The OsWRKY63-OsWRKY76-OsDREB1B module regulates chilling tolerance in rice. Plant J. 112, 383–398 (2022).
Article CAS PubMed Google Scholar
Eremina, M. et al. Hormonal control of cold stress responses in plants. Cell Mol. Life Sci. 73, 797–810 (2016).
Article CAS PubMed Google Scholar
Du, H. et al. A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice. J. Exp. Bot. 63, 6467–6480 (2012).
Article CAS PubMed PubMed Central Google Scholar
Lv, Y. et al. New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome-wide association analysis. Plant Cell Environ. 39, 556–570 (2016).
Article CAS PubMed Google Scholar
Zhao, Y. Auxin biosynthesis and its role in plant development. Annu. Rev. Plant Biol. 61, 49–64 (2010).
Article CAS PubMed PubMed Central Google Scholar
Zhao, Y. Essential roles of local auxin biosynthesis in plant development and in adaptation to environmental changes. Annu. Rev. Plant Biol. 69, 417–435 (2018).
Article CAS PubMed Google Scholar
Li, Y., Han, S. & Qi, Y. Advances in structure and function of auxin response factor in plants. J. Integr. Plant Biol. 65, 617–632 (2023).
Article CAS PubMed Google Scholar
Cui, X. & Cao, X. Epigenetic regulation and functional exaptation of transposable elements in higher plants. Curr. Opin. Plant Biol. 21C, 83–88 (2014).
Article Google Scholar
The 3,000 rice genomes project. The 3,000 rice genomes project. Gigascience 3, 7 (2014).
Domagalska, M. & Leyser, O. Signal integration in the control of shoot branching. Nat. Rev. Mol. Cell Biol. 12, 211–221 (2011).
Article CAS PubMed Google Scholar
Qu, R. et al. Genome-edited ATP BINDING CASSETTE B1 transporter SD8 knockouts show optimized rice architecture without yield penalty. Plant Commun 3, 100347 (2022).
Article CAS PubMed PubMed Central Google Scholar
Zhou, L. et al. ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice. J. Integr. Plant Biol. 65, 1408–1422 (2023).
Article CAS PubMed Google Scholar
Zhang, Y., Berman, A. & Shani, E. Plant hormone transport and localization: signaling molecules on the move. Annu. Rev. Plant Biol. 22, 453–479 (2023).
Article Google Scholar
Swarup, R. & Bhosale, R. Developmental roles of AUX1/LAX auxin influx carriers in plants. Front. Plant Sci. 28, 1306 (2019).
Article Google Scholar
Mellor, N. et al. Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux. Plant Cell 34, 2309–2327 (2022).
Article PubMed PubMed Central Google Scholar
Wu, B. et al. Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice. Nat. Genet. 55, 1381–1389 (2023).
Article CAS PubMed Google Scholar
Eichten, S. et al. Spreading of heterochromatin is limited to specific families of maize retrotransposons. PLoS Genet 8, e1003127 (2012).
Article CAS PubMed PubMed Central Google Scholar
Wang, L., Guo, Y. & Yang, S. Designed breeding for adaptation of crops to environmental abiotic stresses. Sci. Sin. Vitae 51, 1424–1434 (2021).
Article Google Scholar
Li, H. et al. Fine-tuning OsCPK18/OsCPK4 activity via genome editing of phosphorylation motif improves rice yield and immunity. Plant Biotechnol. J. 20, 2258–2271 (2022).
Article CAS PubMed PubMed Central Google Scholar
Kawahara, Y. et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice 6, 4 (2013).
Article PubMed PubMed Central Google Scholar
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Article CAS PubMed PubMed Central Google Scholar
Simon, A., Theodor, P. P. & Wolfgang, H. HTSeq-a python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Article Google Scholar
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 550 (2014).
Article PubMed PubMed Central Google Scholar
Zhao, L. et al. Integrative analysis of reference epigenomes in 20 rice varieties. Nat. Commun. 11, 2658 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, W. et al. High-resolution mapping of open chromatin in the rice genome. Genome Res. 22, 151–162 (2012).
Article CAS PubMed PubMed Central Google Scholar
Guo, Z. et al. Global epigenomic analysis indicates that epialleles contribute to allele-specific expression via allele-specific histone modifications in hybrid rice. BMC Genomics 16, 232 (2015).
Article PubMed PubMed Central Google Scholar
Liu, H. et al. A convenient method for simultaneous quantification of multiple phytohormones and metabolites: application in study of rice-bacterium interaction. Plant Methods 8, 2 (2012).
Article CAS PubMed PubMed Central Google Scholar
Jiang, Y. et al. Isolation and transformation of rice protoplasts. Bio-Protoc 101, e1010125 (2018).
Google Scholar
Kahle, D. & Wickham, H. ggmap: spatial visualization with ggplot2. R. J. 5, 144–161 (2013).
Article Google Scholar
Librado, P. & Rozas, J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452 (2009).
Article CAS PubMed Google Scholar
Browning, B. L., Zhou, Y. & Browning, S. R. A one-penny imputed genome from next-generation reference panels. Am. J. Hum. Genet. 103, 338–348 (2018).
Article CAS PubMed PubMed Central Google Scholar
Garrison, E. et al. A spectrum of free software tools for processing the VCF variant call format: vcflib, bio-vcf, cyvcf2, hts-nim and slivar. PLoS Comput. Biol. 18, e1009123 (2022).
Article CAS PubMed PubMed Central Google Scholar
Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank Dr. Hongzhao Yuan (Institute of Subtropical Agriculture, Chinese Academy of Sciences) for help with deuterium stable isotope determination assays, Dr. Liwei Xu (Institute of Subtropical Agriculture, Chinese Academy of Sciences) for help with auxin determination assays, Dr. Can Peng(Institute of Subtropical Agriculture, Chinese Academy of Sciences) for help with the subcellular localization assay, Prof. Wallace A. Cowling (The University of Western Australia) for providing valuable comments on the manuscript, Prof. Yonglong Chen and Dr. Guanghui Liu (Southern University of Science and Technology) for help with the Xenopus oocytes transport assays, Prof. Kabin Xie (Huazhong Agriculture University) for providing osmap1 rice mutants. This research was supported by National Natural Science Foundation of China (32071995, Recipient: D. M.), Natural Science Foundation of Hunan Province for Distinguished Young scholars (2021J110041, Recipient: D. M.), The Science and Technology Innovation Program of Hunan Province (2023NK1010, Recipient: W. T.), Hainan Seed Industry Laboratory and China National Seed Group (B23YQ1517, Recipient: D. M.), and Research and Development of Technology for Resource Utilization of Biogas Liquid Based on Integrated Crop-Livestock Systems (NCTIP-XDB18).

Author information

These authors contributed equally: Yanchun Cui, Lifang Huang, Peng Liu, and Xiaodong Wang.

Authors and Affiliations

Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China
Yanchun Cui, Lifang Huang, Peng Liu, Xiaodong Wang, Xuan Huang, Xiaojie Hu, Zhankun He, Yuqi Xia, Zebang Li, Caiyan Chen & Donghai Mao
University of Chinese Academy of Sciences, Beijing, China
Xiaodong Wang, Xuan Huang, Yuqi Xia, Zebang Li, Caiyan Chen & Donghai Mao
Yazhou Bay National Laboratory, Sanya, China
Bi Wu & Yongzhong Xing
State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha, China
Yongjun Tan & Wenbang Tang
UWA School of Agriculture and Environment, The University of Western Australia, Perth, Australia
Xiaojie Hu
State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization, CIC-MCP, Nanjing Agricultural University, Nanjing, China
Wenli Zhang
Yuelushan Laboratory, Changsha, China
Donghai Mao

Authors

Yanchun Cui
View author publications
Search author on:PubMed Google Scholar
Lifang Huang
View author publications
Search author on:PubMed Google Scholar
Peng Liu
View author publications
Search author on:PubMed Google Scholar
Xiaodong Wang
View author publications
Search author on:PubMed Google Scholar
Bi Wu
View author publications
Search author on:PubMed Google Scholar
Yongjun Tan
View author publications
Search author on:PubMed Google Scholar
Xuan Huang
View author publications
Search author on:PubMed Google Scholar
Xiaojie Hu
View author publications
Search author on:PubMed Google Scholar
Zhankun He
View author publications
Search author on:PubMed Google Scholar
Yuqi Xia
View author publications
Search author on:PubMed Google Scholar
Zebang Li
View author publications
Search author on:PubMed Google Scholar
Wenli Zhang
View author publications
Search author on:PubMed Google Scholar
Wenbang Tang
View author publications
Search author on:PubMed Google Scholar
Yongzhong Xing
View author publications
Search author on:PubMed Google Scholar
Caiyan Chen
View author publications
Search author on:PubMed Google Scholar
Donghai Mao
View author publications
Search author on:PubMed Google Scholar

Contributions

D.M. conceived and supervised the project. Y.C., P.L., and D.M. designed the experiments. Y. C. carried out the experiments, including the fine mapping of the HAN2 locus, collection of chilling-tolerant phenotypes at the booting stage, DNA methylation analysis, yeast transport experiment, protoplast transformation experiment, feeding of d₅-IAA to rice leaves assay. L.H., and Z.L. carried out the experiments, including GUS histochemical staining and fluorescence immunocytochemistry, collection of chilling-tolerant phenotypes in rice seedlings, seedling auxin and trehalose measurements, analysis the expression of OsMAP1, OsARF24, and OsYUC8 during the seedling stage, yuc8 mutant mature leaf d₅-trp incubation experiment and endogenous IAA measurement. P.L., and X.W. carried out the experiments, including luciferase transient transcriptional activity assay, transport activity assay in Xenopus oocytes, Y1H assays, EMSA, ChIP-assays. P.L. completed the detection of the distribution of OsARF24 free and compound proteins using denaturing and non-denaturing immunoblotting. B.W., Y.T., and Y.C. completed the population genetic analysis of HAN2. Z.H., and Xuan H. completed the molecular marker identification of the HAN2 population materials and the molecular marker identification of retrotransposons. Xiaojie. H. analyzed the chilling tolerance differences of HAN2 in different varieties of indica and japonica rice. Yuqi X. conducted subcellular localization experiments and the genotyping and phenotyping of osarf24. W.Z. completed the analysis of methylation and histone methylation modifications. W.T. provided rice germplasm materials and field experiment guidance. Yongzhong X., and C.C. provided theoretical contributions to the project. D.M., Y.C., and P.L. wrote the manuscript. All the authors read and approved the manuscript.

Corresponding author

Correspondence to Donghai Mao.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Reporting Summary

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Peer Review file

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Cui, Y., Huang, L., Liu, P. et al. Suppressing an auxin efflux transporter enhances rice adaptation to temperate habitats. Nat Commun 16, 4100 (2025). https://doi.org/10.1038/s41467-025-59449-z

Download citation

Received: 12 August 2024
Accepted: 23 April 2025
Published: 02 May 2025
DOI: https://doi.org/10.1038/s41467-025-59449-z