Citrus is one of the most important fruit crop genera in the world, but many Citrus species are vulnerable to cold stress. Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, holds great potential for identifying valuable metabolites that are critical for cold tolerance in Citrus. However, the metabolic changes and underlying mechanisms that regulate Ichang papeda cold tolerance remain largely unknown. In this study, we compared the metabolomes and transcriptomes of Ichang papeda and HB pummelo (Citrus grandis ‘Hirado Buntan’, a cold-sensitive species) to explore the critical metabolites and genes responsible for cold tolerance. Metabolomic analyses led to the identification of common and genotype-specific metabolites, consistent with transcriptomic alterations. Compared to HB pummelo under cold stress, Ichang papeda accumulated more sugars, flavonoids, and unsaturated fatty acids, which are well-characterized metabolites involved in stress responses. Interestingly, sphingosine and chlorogenic acid substantially accumulated only in Ichang papeda. Knockdown of CiSPT (C. ichangensis serine palmitoyltransferase) and CiHCT2 (C. ichangensis hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase2), two genes involved in sphingosine and chlorogenic acid biosynthesis, dramatically decreased endogenous sphingosine and chlorogenic acid levels, respectively. This reduction in sphingosine and chlorogenic acid notably compromised the cold tolerance of Ichang papeda, whereas exogenous application of these metabolites increased plant cold tolerance. Taken together, our findings indicate that greater accumulation of a spectrum of metabolites, particularly sphingosine and chlorogenic acid, promotes cold tolerance in cold-tolerant citrus species. These findings broaden our understanding of plant metabolic alterations in response to cold stress and provide valuable targets that can be manipulated to improve Citrus cold tolerance.

Plants subjected to cold stress have been observed to accumulate proline, but the underlying regulatory mechanism remains to be elucidated. In this study, we identified a pyrroline-5-carboxylate synthetase (P5CS)-encoding gene (CtrP5CS1) from trifoliate orange (Citrus trifoliata L.), a cold-hardy citrus species, as a critical gene for cold-induced proline accumulation. CtrTGA2 bound directly to the TGACG motif of the CtrP5CS1 promoter and activated its expression. Moreover, CtrTGA2 functioned positively in cold tolerance via modulation of proline synthesis by regulating CtrP5CS1 expression. Up-regulation of CtrP5CS1 and CtrTGA2 under cold stress was dependent on salicylic acid (SA) biosynthesis. CtrTGA2 directly regulated the expression of CtrICS1, a gene encoding isochorismate synthase (ICS) involved in SA biosynthesis, forming a positive feedback loop to intensify the CtrTGA2-mediated transcriptional activation of CtrP5CS1. The cold-induced SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES3 (CtrNPR3) interacted with CtrTGA2 to inhibit its transcriptional activation activity; however, the inhibition was released by SA. Our results uncover the CtrTGA2-CtrP5CS1/CtrICS1 regulatory module that orchestrates the SA signal to regulate proline synthesis, giving important insights into the transcriptional mechanism underlying proline accumulation in plants under cold stress.

GRAS, termed after GAI (gibberellic acid insensitive), RGA (repressor of GA1), and SCR (scarecrow), is a plant-specific transcription factor crucial for plant development and stress response. However, understanding of the functions played by the GRAS members and their target genes in citrus is limited. In this study, we identified a cold stress-responsive GRAS gene from Poncirus trifoliate, designated as PtrPAT1, by yeast one-hybrid library screening using the promoter of PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrPAT1, belonging to the PAT1 subfamily, was localized in the nucleus and plasma membrane, exhibited transactivation activity and showed a remarkable upregulation under cold stress. Overexpression of PtrPAT1 elevated BADH activity, increased glycine betaine (GB) accumulation and conferred enhanced cold tolerance in transgenic tobacco plants compared with wild type, while downregulation in trifoliate orange by virus-induced gene silencing (VIGS) resulted in opposite trends. Furthermore, the activities of two antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were significantly increased in the overexpression plants, but remarkably decreased in the VIGS line, consistent with accumulation patterns of the reactive oxygen species (ROS). PtrPAT1 was demonstrated to interact with and activate the PtrBADH-l promoter through the putative PAT1-binding motif with the core sequence of TTTCATGT, indicating that PtrBADH-l is a target gene of PtrPAT1. Taken together, these results demonstrate that PtrPAT1 positively affects cold tolerance through the regulation of GB biosynthesis by modulating PtrBADH-l expression.

Spermidine (Spd) is one of the predominant polyamines in higher plants and plays a crucial role in combating various abiotic stresses. However, the molecular functions and underlying regulatory mechanisms associated with plant Spd synthase (SPDS) genes in cold tolerance remain poorly understood. In this study, cold treatment markedly induced Spd accumulation and enhanced SPDS activity in Ichang papeda (Citrus ichangensis), a cold-hardy plant in Citrus genus. Exogenous Spd supply led to dramatically improved cold tolerance. Two SPDS genes (CiSPDS1 and CiSPDS2) were identified in Ichang papeda, but only CiSPDS1 was substantially upregulated by cold. Overexpressing of CiSPDS1 in both tobacco (Nicotiana tabacum) and lemon (Citrus limon), a cold-sensitive Citrus species, promoted Spd synthesis and enhanced cold tolerance in the transgenic plants. In contrast, knockdown of CiSPDS1 in Ichang papeda by virus-induced gene silencing (VIGS) repressed Spd synthesis and greatly impaired the cold tolerance, which was restored by exogenous replenishment of Spd. In addition, we demonstrated that WRKY27 of Ichang papeda (CiWRKY27) directly bound to and activated the CiSPDS1 promoter through interacting with a W-box cis-acting element. Meanwhile, VIGS-mediated silencing of CiWRKY27 resulted in marked reduction of CiSPDS1 transcript levels and Spd contents and significantly impaired the cold tolerance in Ichang papeda. Taken together, this study illustrated the role of CiSPDS1 in cold tolerance and identified it as a direct target of CiWRKY27. These findings provide insight into the regulatory mechanism by which the molecular module CiWRKY27-CiSPDS1 regulates Spd accumulation for modulation of cold tolerance.

Fruit ripening is a complex, tightly regulated process that affects fruit flavor, nutritional value, and shelf life. Citrus is a typical non-climacteric fruit, as citrus fruits ripen independently of ethylene. However, the precise regulatory mechanism underlying this process remains to be elucidated. To dissect the epigenetic and transcriptional basis of citrus ripening, we performed integrative analyses of genome-wide histone modifications (H3K27ac, H3K4me3, and H3K27me3), chromatin accessibility, and transcriptome profiles throughout fruit ripening in Citrus reticulata. We constructed a transcriptional regulatory network encompassing 52 key transcription factors that orchestrate citrus fruit ripening. Further, we identified the key role of abscisic acid (ABA) in modulating sucrose and citric acid metabolism and uncovered the underlying transcriptional and epigenetic regulatory network during fruit ripening in C. reticulata. The findings demonstrate that CrHSFA6B and CrbZIP5b interact with the promoters of CrACO1 (Aconitase 1) and CrACO3 and CrSPSF1 (Sucrose Phosphate Synthase F1), regulating their expression to affect the sucrose and citric acid metabolism and fruit ripening. This study provides insights into the epigenomic dynamics of citrus fruit ripening and identifies a putative regulatory cascade centered on ABA, CrbZIP5b, and CrHSFA6B that modulates key processes in citrus ripening.

Plant melatonin is widely recognized as a pleiotropic regulator. As a growth-regulating hormone, it extensively participates in various growth and developmental processes and has significant functions in stress responses and disease resistance. Plant melatonin is synthesized primarily through the catalytic actions of five enzymes: TDC (tryptophan decarboxylase), T5H (tryptamine-5-hydroxylase), SNAT (serotonin N-acetyltransferase), ASMT (N-acetylserotonin methyltransferase), and COMT (caffeic acid-O-methyltransferase). There are multiple genes for each of these five enzymes in citrus genomes, however, with the exception of COMT5—whose function has recently been elucidated—and SNAT, which has only been preliminarily identified, the remaining genes have not been unequivocally characterized or functionally annotated. Hence, we carried out a genome-wide analysis of melatonin biosynthesis enzyme-related gene families in trifoliate orange (Poncirus trifoliata), one of the most common citrus rootstock varieties. Through bioinformatics approaches, we identified 96 gene family members encoding melatonin biosynthetic enzymes and characterized their protein sequence properties, phylogenetic relationships, gene structures, chromosomal distributions, and promoter cis-acting elements. Furthermore, by analyzing expression patterns in different tissues and under various stresses, we identified multiple stress-responsive melatonin synthase genes. These genes likely participate in melatonin synthesis under adverse conditions, thereby enhancing stress adaptation. Specifically, PtCOMT5, PtASMT11, and PtTDC9 were significantly induced by low temperature; PtSNAT1, PtSNAT14, PtSNAT18, and PtTDC10 were markedly responsive to drought; and PtASMT15, PtSNAT15, PtASMT16, and PtSNAT3 were strongly induced by ABA. Among them, PtASMT23 expression was induced up to 120-fold under low temperature, while PtSNAT18 showed over 100-fold upregulation under dehydration treatment. These findings strongly suggest that PtASMT23 and PtSNAT18 play critical roles in regulating melatonin biosynthesis in response to cold and drought stress, respectively. Collectively, these findings pinpoint novel genetic targets for enhancing stress resilience in citrus breeding programs and lay the foundation for the functional characterization of specific melatonin biosynthesis pathway gene family members in citrus and other horticultural crop species.