Cui Xiao

Citrus fruits are widely cultivated all over the world. Due to climatic conditions, citrus fruits are frequently exposed to periodic low temperatures, which poses a serious threat to their yield and quality. Cold not only restricts plant growth and deteriorates fruit quality but also leads to fruit abscission and tree mortality, posing severe constraints on large-scale citrus production. The TIFY family gene plays crucial roles in plant development and stress adaptation. However, the genome-wide identification and functional analysis of TIFY genes in cold stress adaptation of citrus plants remain largely unexplored. Here, we performed a systematic genome-wide analysis of the TIFY family in sweet orange (Citrus sinensis (L.) Osbeck) and identified 14 CsTIFY members. We conducted a comprehensive study on the protein characteristics, phylogenetic relationships, gene structure, chromosome distribution, promoter cis-acting elements, and subcellular localization of these genes. Phylogenetic analysis classified the CsTIFYs into ZML (ZML1–ZML4), JAZ (JAZ1–JAZ7), PPD (JAZ8, JAZ9), and TIFY (TIFY1) subfamilies, and they are distributed on seven chromosomes. Collinearity analysis revealed that segmental duplication is the primary driver for CsTIFY family expansion. Expression profiling under cold stress identified JAZ1, JAZ2, and JAZ3 as the most cold-inducible members. All three CsTIFY proteins are targeted to the nucleus, as confirmed by subcellular localization analysis. Overexpression of JAZ1, JAZ2, or JAZ3 in citrus calli significantly enhanced cold sensibility. Collectively, this study elucidates the gene function of CsTIFYs under cold stress and provides new insight for molecular breeding of cold-tolerant citrus varieties.

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.

Flavor formation in citrus fruit is governed by complex and dynamic changes in primary and secondary metabolism during development and ripening. Here, we integrated metabolomic, hormonal, and transcriptomic analyses to elucidate the regulatory landscape underlying soluble sugar and organic acid metabolism in ‘Kiyomi’ citrus fruit. A total of 1679 metabolites were identified, revealing stage-specific reprogramming of metabolic pathways, including a sharp decline in citric acid after 90 days after flowering (DAF) and peak accumulation of sucrose at 180 DAF. Hormonal profiling showed that abscisic acid (ABA) progressively accumulated from 120 to 210 DAF, while 1-aminocyclopropane-1-carboxylic acid (ACC) peaked at 120 DAF and declined thereafter, suggesting distinct and temporally coordinated roles in ripening regulation. Transcriptomic profiling uncovered widespread temporal shifts in gene expression, with the most pronounced changes occurring between 180 and 210 DAF. Co-expression network analysis identified gene modules associated with sugar and acid accumulation, and highlighted transcription factors from the ERF, MYB, NAC, and HSF families as candidate regulators of ripening-related metabolic transitions. These findings provide a comprehensive framework for understanding the coordinated molecular and metabolic programs underlying flavor development in non-climacteric citrus fruit and offer candidate genes for the genetic improvement of fruit quality.

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5′-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.

Cold is one of the major limiting factors for citrus production, particularly extreme cold waves. Therefore, it is of great importance to develop cold-tolerant varieties and clarify their cold tolerance mechanisms in citrus breeding. In this study, comparative transcriptomic and physiological analyses were performed to dissect the cold tolerance mechanism of Guijing2501 (GJ2501), a new satsuma mandarin (Citrus unshiu) variety with about 1 °C lower LT50 (the median lethal temperature) relative to Guijing (GJ). The physiological analysis results revealed that GJ2501 is more cold-tolerant with less photoinhibition, PSII photodamage, and MDA accumulation, but higher POD activity than GJ under cold stress. Comparative transcriptomic analysis identified 4200 DEGs between GJ and GJ2501, as well as 4884 and 5580 up-regulated DEGs, and 5288 and 5862 down-regulated DEGs in response to cold stress in GJ and GJ2501, respectively. “Photosynthesis, light harvesting” and “photosystem” were the specific and most significantly enriched GO terms in GJ2501 in response to cold stress. Two CuELIP1 genes (encoding early light-induced proteins) related to the elimination of PSII photodamage and photoinhibition were remarkably up-regulated (by about 1000-fold) by cold stress in GJ2501 as indicated by RT-qPCR verification. Overexpression of CuELIP1 from GJ2501 in transgenic Arabidopsis protected PSII against photoinhibition under cold stress. Taken together, the cold tolerance of GJ2501 may be ascribed to its higher photoprotective capacity under cold stress.

Objectives: Pollution exposure is associated with several dermatological conditions including acne, atopic dermatitis, and psoriasis. Antimicrobial peptides (AMPs) are key effectors of innate defense, and some AMPs are involved in inflammatory skin conditions. In this study, we aimed to characterize expression changes of human AMPs under different in-vitro pollution exposures. Methods: RNA-seq profiling was conducted on normal human primary epidermal keratinocytes (NHEK) treated with either a vehicle control, or benzo[a]pyrene (BaP) and on pigmented living skin equivalent models (pLSE) treated with either a vehicle control, ozone, or vehicle exhaust. Differential expressed genes (DEGs) were identified with R scripts. DEGs of PM2.5 were obtained from the literature and the GEO database. Also, 180 human AMP genes were obtained from a UDAMP database. UpSetPlot was used to plot DEGs overlaps. MetaVolcano was used to identify frequently changed AMPs. Results: We used in-house and published transcriptome profiles to identify AMP genes that displayed altered expression under in-vitro pollution exposure. Of the 180 AMP genes under investigation, 37 showed significant changes in expression in at least 1 of the 5 experiments. Using MetaVolcano, 13 AMP genes were identified to be frequently and consistently changed. Several AMPs associated with inflammation and skin diseases were frequently upregulated, including S100A8, S100A9, LCN2, HBD3, RNASE7, and CXCL1. Only 3 frequently downregulated AMP genes were identified, including CXCL14, which is reported to be a noninflammatory AMP that is highly expressed in healthy skin and is downregulated in skin diseases. Conclusions: The data sets suggest that expression of both proinflammatory and homeostatic AMPs can be perturbed by pollution. These findings provide new clues to explain how pollution affects skin innate defense, host–microbe interactions and contributes to abnormal skin conditions. Normalizing aberrant AMP expression may be a potential approach to treat pollution associated skin disorders in the future.

Background: Cold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca²⁺-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus? Results: Eight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin ‘Guijing2501’ (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content. Conclusions: In this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.