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Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume

  • # Authors contributed equally: Haolin Liu, Lihong Hao

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  • Received: 22 October 2023
    Revised: 19 February 2024
    Accepted: 27 February 2024
    Published online: 03 April 2024
    Tropical Plants  3 Article number: e010 (2024)  |  Cite this article
  • For the first time, six PmCBL genes had been comprehensively and systematically identified in P. mume.

    The PmCBL gene family responded to cold stress, and PmCBL1/3/5/6 were identified as potential key genes involved in regulating cold tolerance in P. mume.

  • The CBL gene family is an important family in the Ca2+ mediated signal transduction pathway in plants and plays a crucial role in plant stress responses and growth development. However, research on the response of members of the Prunus mume CBL gene family to low temperature stress remains scarce. In this study, we systematically analyzed the protein physicochemical properties, chromosome localization, phylogenetic evolution, gene structure, conserved domains, cis-acting elements, and gene expression patterns in response to low temperature stress of members of the P. mume CBL gene family using bioinformatics tools. Six PmCBL gene family members were identified in the P. mume genome. Phylogenetic trees were constructed, revealing three subfamilies named Group I, Group II, and Group III. In the P. mume gene family, PmCBL4 and PmCBL5 were paralogous genes. The members of the P. mume CBL gene family were unevenly distributed on three chromosomes. The CBL encoding protein, the number of isoelectric points (pI), the number of introns and exons of the six gene families were different. Analysis of the upstream 700 bp promoter sequences of the P. mume CBL gene family revealed the presence of various types of cis-acting elements involved in non-biological stress responses. Among the six identified genes, each gene exhibited different expression patterns in response to low temperature. Among them, the up-regulated expression of PmCBL5 was the largest, and the expression of PmCBL1, PmCBL3 and PmCBL5 showed the up-regulated trend. These results indicated that PmCBL1, PmCBL3, PmCBL5, and PmCBL6 were key genes involved in the response of P. mume to low temperature stress. This study provided comprehensive and systematic analysis of the P. mume CBL gene family members and identified key genes involved in the response to low temperature stress, thereby providing genetic resources for molecular breeding programs aimed at enhancing cold resistance in P. mume.
    Graphical Abstract
  • As a major staple crop, today maize accounts for approximately 40% of total worldwide cereal production (http://faostat.fao.org/). Since its domestication ~9,000 years ago from a subgroup of teosinte (Zea mays ssp. parviglumis) in the tropical lowlands of southwest Mexico[1], its cultivating area has greatly expanded, covering most of the world[2]. Human's breeding and utilization of maize have gone through several stages, from landraces, open-pollinated varieties (OPVs), double-cross hybrids (1930s-1950s) and since the middle 1950s, single-cross hybrids. Nowadays, global maize production is mostly provided by single-cross hybrids, which exhibit higher-yielding and better stress tolerance than OPVs and double-cross hybrids[3].

    Besides its agronomic importance, maize has also been used as a model plant species for genetic studies due to its out-crossing habit, large quantities of seeds produced and the availability of diverse germplasm. The abundant mutants of maize facilitated the development of the first genetic and cytogenetic maps of plants, and made it an ideal plant species to identify regulators of developmental processes[46]. Although initially lagging behind other model plant species (such as Arabidopsis and rice) in multi-omics research, the recent rapid development in sequencing and transformation technologies, and various new tools (such as CRISPR technologies, double haploids etc.) are repositioning maize research at the frontiers of plant research, and surely, it will continue to reveal fundamental insights into plant biology, as well as to accelerate molecular breeding for this vitally important crop[7, 8].

    During domestication from teosinte to maize, a number of distinguishing morphological and physiological changes occurred, including increased apical dominance, reduced glumes, suppression of ear prolificacy, increase in kernel row number, loss of seed shattering, nutritional changes etc.[9] (Fig. 1). At the genomic level, genome-wide genetic diversity was reduced due to a population bottleneck effect, accompanied by directional selection at specific genomic regions underlying agronomically important traits. Over a century ago, Beadle initially proposed that four or five genes or blocks of genes might be responsible for much of the phenotypic changes between maize and teosinte[10,11]. Later studies by Doebley et al. used teosinte–maize F2 populations to dissect several quantitative trait loci (QTL) to the responsible genes (such as tb1 and tga1)[12,13]. On the other hand, based on analysis of single-nucleotide polymorphisms (SNPs) in 774 genes, Wright et al.[14] estimated that 2%−4% of maize genes (~800−1,700 genes genome-wide) were selected during maize domestication and subsequent improvement. Taking advantage of the next-generation sequencing (NGS) technologies, Hufford et al.[15] conducted resequencing analysis of a set of wild relatives, landraces and improved maize varieties, and identified ~500 selective genomic regions during maize domestication. In a recent study, Xu et al.[16] conducted a genome-wide survey of 982 maize inbred lines and 190 teosinte accession. They identified 394 domestication sweeps and 360 adaptation sweeps. Collectively, these studies suggest that maize domestication likely involved hundreds of genomic regions. Nevertheless, much fewer domestication genes have been functionally studied so far.

    Figure 1.  Main traits of maize involved in domestication and improvement.

    During maize domestication, a most profound morphological change is an increase in apical dominance, transforming a multi-branched plant architecture in teosinte to a single stalked plant (terminated by a tassel) in maize. The tillers and long branches of teosinte are terminated by tassels and bear many small ears. Similarly, the single maize stalk bears few ears and is terminated by a tassel[9,12,17]. A series of landmark studies by Doebley et al. elegantly demonstrated that tb1, which encodes a TCP transcription factor, is responsible for this transformation[18, 19]. Later studies showed that insertion of a Hopscotch transposon located ~60 kb upstream of tb1 enhances the expression of tb1 in maize, thereby repressing branch outgrowth[20, 21]. Through ChIP-seq and RNA-seq analyses, Dong et al.[22] demonstrated that tb1 acts to regulate multiple phytohormone signaling pathways (gibberellins, abscisic acid and jasmonic acid) and sugar sensing. Moreover, several other domestication loci, including teosinte glume architecture1 (tga1), prol1.1/grassy tillers1, were identified as its putative targets. Elucidating the precise regulatory mechanisms of these loci and signaling pathways will be an interesting and rewarding area of future research. Also worth noting, studies showed that tb1 and its homologous genes in Arabidopsis (Branched1 or BRC1) and rice (FINE CULM1 or FC1) play a conserved role in repressing the outgrowth of axillary branches in both dicotyledon and monocotyledon plants[23, 24].

    Teosinte ears possess two ranks of fruitcase-enclosed kernels, while maize produces hundreds of naked kernels on the ear[13]. tga1, which encodes a squamosa-promoter binding protein (SBP) transcription factor, underlies this transformation[25]. It has been shown that a de novo mutation occurred during maize domestication, causing a single amino acid substitution (Lys to Asn) in the TGA1 protein, altering its binding activity to its target genes, including a group of MADS-box genes that regulate glume identity[26].

    Prolificacy, the number of ears per plants, is also a domestication trait. It has been shown that grassy tillers 1 (gt1), which encodes an HD-ZIP I transcription factor, suppresses prolificacy by promoting lateral bud dormancy and suppressing elongation of the later ear branches[27]. The expression of gt1 is induced by shading and requires the activity of tb1, suggesting that gt1 acts downstream of tb1 to mediate the suppressed branching activity in response to shade. Later studies mapped a large effect QTL for prolificacy (prol1.1) to a 2.7 kb 'causative region' upstream of the gt1gene[28]. In addition, a recent study identified a new QTL, qEN7 (for ear number on chromosome 7). Zm00001d020683, which encodes a putative INDETERMINATE DOMAIN (IDD) transcription factor, was identified as the likely candidate gene based on its expression pattern and signature of selection during maize improvement[29]. However, its functionality and regulatory relationship with tb1 and gt1 remain to be elucidated.

    Smaller leaf angle and thus more compact plant architecture is a desired trait for modern maize varieties. Tian et al.[30] used a maize-teosinte BC2S3 population and cloned two QTLs (Upright Plant Architecture1 and 2 [UPA1 and UPA2]) that regulate leaf angle. Interestingly, the authors showed that the functional variant of UPA2 is a 2-bp InDel located 9.5 kb upstream of ZmRAVL1, which encodes a B3 domain transcription factor. The 2-bp Indel flanks the binding site of the transcription factor Drooping Leaf1 (DRL1)[31], which represses ZmRAVL1 expression through interacting with Liguleless1 (LG1), a SBP-box transcription factor essential for leaf ligule and auricle development[32]. UPA1 encodes brassinosteroid C-6 oxidase1 (brd1), a key enzyme for biosynthesis of active brassinolide (BR). The teosinte-derived allele of UPA2 binds DRL1 more strongly, leading to lower expression of ZmRAVL1 and thus, lower expression of brd1 and BR levels, and ultimately smaller leaf angle. Notably, the authors demonstrated that the teosinte-derived allele of UPA2 confers enhanced yields under high planting densities when introgressed into modern maize varieties[30, 33].

    Maize plants exhibit salient vegetative phase change, which marks the vegetative transition from the juvenile stage to the adult stage, characterized by several changes in maize leaves produced before and after the transition, such as production of leaf epicuticular wax and epidermal hairs. Previous studies reported that Glossy15 (Gl15), which encodes an AP2-like transcription factor, promotes juvenile leaf identity and suppressing adult leaf identity. Ectopic overexpression of Gl15 causes delayed vegetative phase change and flowering, while loss-of-function gl15 mutant displayed earlier vegetative phase change[34]. In another study, Gl15 was identified as a major QTL (qVT9-1) controlling the difference in the vegetative transition between maize and teosinte. Further, it was shown that a pre-existing low-frequency standing variation, SNP2154-G, was selected during domestication and likely represents the causal variation underlying differential expression of Gl15, and thus the difference in the vegetative transition between maize and teosinte[35].

    A number of studies documented evidence that tassels replace upper ears1 (tru1) is a key regulator of the conversion of the male terminal lateral inflorescence (tassel) in teosinte to a female terminal inflorescence (ear) in maize. tru1 encodes a BTB/POZ ankyrin repeat domain protein, and it is directly targeted by tb1, suggesting their close regulatory relationship[36]. In addition, a number of regulators of maize inflorescence morphology, were also shown as selective targets during maize domestication, including ramosa1 (ra1)[37, 38], which encodes a putative transcription factor repressing inflorescence (the ear and tassel) branching, Zea Agamous-like1 (zagl1)[39], which encodes a MADS-box transcription factor regulating flowering time and ear size, Zea floricaula leafy2 (zfl2, homologue of Arabidopsis Leafy)[40, 41], which likely regulates ear rank number, and barren inflorescence2 (bif2, ortholog of the Arabidopsis serine/threonine kinase PINOID)[42, 43], which regulates the formation of spikelet pair meristems and branch meristems on the tassel. The detailed regulatory networks of these key regulators of maize inflorescence still remain to be further elucidated.

    Kernel row number (KRN) and kernel weight are two important determinants of maize yield. A number of domestication genes modulating KRN and kernel weight have been identified and cloned, including KRN1, KRN2, KRN4 and qHKW1. KRN4 was mapped to a 3-kb regulatory region located ~60 kb downstream of Unbranched3 (UB3), which encodes a SBP transcription factor and negatively regulates KRN through imparting on multiple hormone signaling pathways (cytokinin, auxin and CLV-WUS)[44, 45]. Studies have also shown that a harbinger TE in the intergenic region and a SNP (S35) in the third exon of UB3 act in an additive fashion to regulate the expression level of UB3 and thus KRN[46].

    KRN1 encodes an AP2 transcription factor that pleiotropically affects plant height, spike density and grain size of maize[47], and is allelic to ids1/Ts6 (indeterminate spikelet 1/Tassel seed 6)[48]. Noteworthy, KRN1 is homologous to the wheat domestication gene Q, a major regulator of spike/spikelet morphology and grain threshability in wheat[49].

    KRN2 encodes a WD40 domain protein and it negatively regulates kernel row number[50]. Selection in a ~700-bp upstream region (containing the 5’UTR) of KRN2 during domestication resulted in reduced expression and thus increased kernel row number. Interestingly, its orthologous gene in rice, OsKRN2, was shown also a selected gene during rice domestication to negatively regulate secondary panicle branches and thus grain number. These observations suggest convergent selection of yield-related genes occurred during parallel domestication of cereal crops.

    qHKW1 is a major QTL for hundred-kernel weight (HKW)[51]. It encodes a CLAVATA1 (CLV1)/BARELY ANY MERISTEM (BAM)-related receptor kinase-like protein positively regulating HKW. A 8.9 Kb insertion in its promoter region was find to enhance its expression, leading to enhanced HKW[52]. In addition, Chen et al.[53] reported cloning of a major QTL for kernel morphology, qKM4.08, which encodes ZmVPS29, a retromer complex component. Sequencing and association analysis revealed that ZmVPS29 was a selective target during maize domestication. They authors also identified two significant polymorphic sites in its promoter region significantly associated with the kernel morphology. Moreover, a strong selective signature was detected in ZmSWEET4c during maize domestication. ZmSWEET4c encodes a hexose transporter protein functioning in sugar transport across the basal endosperm transfer cell layer (BETL) during seed filling[54]. The favorable alleles of these genes could serve as valuable targets for genetic improvement of maize yield.

    In a recent effort to more systematically analyze teosinte alleles that could contribute to yield potential of maize, Wang et al.[55] constructed four backcrossed maize-teosinte recombinant inbred line (RIL) populations and conducted detailed phenotyping of 26 agronomic traits under five environmental conditions. They identified 71 QTL associated with 24 plant architecture and yield related traits through inclusive composite interval mapping. Interestingly, they identified Zm00001eb352570 and Zm00001eb352580, both encode ethylene-responsive transcription factors, as two key candidate genes regulating ear height and the ratio of ear to plant height. Chen et al.[56] constructed a teosinte nested association mapping (TeoNAM) population, and performed joint-linkage mapping and GWAS analyses of 22 domestication and agronomic traits. They identified the maize homologue of PROSTRATE GROWTH1, a rice domestication gene controlling the switch from prostrate to erect growth, is also a QTL associated with tillering in teosinte and maize. Additionally, they also detected multiple QTL for days-to-anthesis (such as ZCN8 and ZmMADS69) and other traits (such as tassel branch number and tillering) that could be exploited for maize improvement. These lines of work highlight again the value of mining the vast amounts of superior alleles hidden in teosinte for future maize genetic improvement.

    Loss of seed shattering was also a key trait of maize domestication, like in other cereals. shattering1 (sh1), which encodes a zinc finger and YABBY domain protein regulating seed shattering. Interesting, sh1 was demonstrated to undergo parallel domestication in several cereals, including rice, maize, sorghum, and foxtail millet[57]. Later studies showed that the foxtail millet sh1 gene represses lignin biosynthesis in the abscission layer, and that an 855-bp Harbinger transposable element insertion in sh1 causes loss of seed shattering in foxtail millet[58].

    In addition to morphological traits, a number of physiological and nutritional related traits have also been selected during maize domestication. Based on survey of the nucleotide diversity, Whitt et al.[59] reported that six genes involved in starch metabolism (ae1, bt2, sh1, sh2, su1 and wx1) are selective targets during maize domestication. Palaisa et al.[60] reported selection of the Y1 gene (encoding a phytoene synthase) for increased nutritional value. Karn et al.[61] identified two, three, and six QTLs for starch, protein and oil respectively and showed that teosinte alleles can be exploited for the improvement of kernel composition traits in modern maize germplasm. Fan et at.[62] reported a strong selection imposed on waxy (wx) in the Chinese waxy maize population. Moreover, a recent exciting study reported the identification of a teosinte-derived allele of teosinte high protein 9 (Thp9) conferring increased protein level and nitrogen utilization efficiency (NUE). It was further shown that Thp9 encodes an asparagine synthetase 4 and that incorrect splicing of Thp9-B73 transcripts in temperate maize varieties is responsible for its diminished expression, and thus reduced NUE and protein content[63].

    Teosintes is known to confer superior disease resistance and adaptation to extreme environments (such as low phosphorus and high salinity). de Lange et al. and Lennon et al.[6466] reported the identification of teosinte-derived QTLs for resistance to gray leaf spot and southern leaf blight in maize. Mano & Omori reported that teosinte-derived QTLs could confer flooding tolerance[67]. Feng et al.[68] identified four teosinte-derived QTL that could improve resistance to Fusarium ear rot (FER) caused by Fusarium verticillioides. Recently, Wang et al.[69] reported a MYB transcription repressor of teosinte origin (ZmMM1) that confers resistance to northern leaf blight (NLB), southern corn rust (SCR) and gray leaf spot (GLS) in maize, while Zhang et al.[70] reported the identification of an elite allele of SNP947-G ZmHKT1 (encoding a sodium transporter) derived from teosinte can effectively improve salt tolerance via exporting Na+ from the above-ground plant parts. Gao et al.[71] reported that ZmSRO1d-R can regulate the balance between crop yield and drought resistance by increasing the guard cells' ROS level, and it underwent selection during maize domestication and breeding. These studies argue for the need of putting more efforts to tapping into the genetic resources hidden in the maize’s wild relatives. The so far cloned genes involved in maize domestication are summarized in Table 1. Notably, the enrichment of transcription factors in the cloned domestication genes highlights a crucial role of transcriptional re-wiring in maize domestication.

    Table 1.  Key domestication genes cloned in maize.
    GenePhenotypeFunctional annotationSelection typeCausative changeReferences
    tb1Plant architectureTCP transcription factorIncreased expression~60 kb upstream of tb1 enhancing expression[1822]
    tga1Hardened fruitcaseSBP-domain transcription factorProtein functionA SNP in exon (K-N)[25, 26]
    gt1Plant architectureHomeodomain leucine zipperIncreased expressionprol1.1 in 2.7 kb upstream of the promoter region increasing expression[27, 28]
    Zm00001d020683Plant architectureINDETERMINATE DOMAIN transcription factorProtein functionUnknown[29]
    UPA1Leaf angleBrassinosteroid C-6 oxidase1Protein functionUnknown[30]
    UPA2Leaf angleB3 domain transcription factorIncreased expressionA 2 bp indel in 9.5 kb upstream of ZmRALV1[30]
    Gl15Vegetative phase changeAP2-like transcription factorAltered expressionSNP2154: a stop codon (G-A)[34, 35]
    tru1Plant architectureBTB/POZ ankyrin repeat proteinIncreased expressionUnknown[36]
    ra1Inflorescence architectureTranscription factorAltered expressionUnknown[37, 38]
    zflPlant architectureTranscription factorAltered expressionUnknown[40, 41]
    UB3Kernel row numberSBP-box transcription factorAltered expressionA TE in the intergenic region;[4446]
    SNP (S35): third exon of UB3
    (A-G) increasing expression of UB3 and KRN
    KRN1/ids1/Ts6Kernel row numberAP2 Transcription factorIncreased expressionUnknown[47, 48]
    KRN2Kernel row numberWD40 domainDecreased expressionUnknown[50]
    qHKW1Kernel row weightCLV1/BAM-related receptor kinase-like proteinIncreased expression8.9 kb insertion upstream of HKW[51, 52]
    ZmVPS29Kernel morphologyA retromer complex componentProtein functionTwo SNPs (S-1830 and S-1558) in the promoter of ZmVPS29[53]
    ZmSWEET4cSeed fillingHexose transporterProtein functionUnknown[54]
    ZmSh1ShatteringA zinc finger and YABBY transcription factorProtein functionUnknown[57, 58]
    Thp9Nutrition qualityAsparagine synthetase 4 enzymeProtein functionA deletion in 10th intron of Thp9 reducing NUE and protein content[63]
    ZmMM1Biotic stressMYB Transcription repressorProtein functionUnknown[69]
    ZmHKT1Abiotic stressA sodium transporterProtein functionSNP947-G: a nonsynonymous variation increasing salt tolerance[70]
    ZmSRO1d-RDrought resistance and productionPolyADP-ribose polymerase and C-terminal RST domainProtein functionThree non-synonymous variants: SNP131 (A44G), SNP134 (V45A) and InDel433[71]
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    After its domestication from its wild progenitor teosinte in southwestern Mexico in the tropics, maize has now become the mostly cultivated crop worldwide owing to its extensive range expansion and adaptation to diverse environmental conditions (such as temperature and day length). A key prerequisite for the spread of maize from tropical to temperate regions is reduced photoperiod sensitivity[72]. It was recently shown that CENTRORADIALIS 8 (ZCN8), an Flowering Locus T (FT) homologue, underlies a major quantitative trait locus (qDTA8) for flowering time[73]. Interestingly, it has been shown that step-wise cis-regulatory changes occurred in ZCN8 during maize domestication and post-domestication expansion. SNP-1245 is a target of selection during early maize domestication for latitudinal adaptation, and after its fixation, selection of InDel-2339 (most likely introgressed from Zea mays ssp. Mexicana) likely contributed to the spread of maize from tropical to temperate regions[74].

    ZCN8 interacts with the basic leucine zipper transcription factor DLF1 (Delayed flowering 1) to form the florigen activation complex (FAC) in maize. Interestingly, DFL1 was found to underlie qLB7-1, a flowering time QTL identified in a BC2S3 population of maize-teosinte. Moreover, it was shown that DLF1 directly activates ZmMADS4 and ZmMADS67 in the shoot apex to promote floral transition[75]. In addition, ZmMADS69 underlies the flowering time QTL qDTA3-2 and encodes a MADS-box transcription factor. It acts to inhibit the expression of ZmRap2.7, thereby relieving its repression on ZCN8 expression and causing earlier flowering. Population genetic analyses showed that DLF1, ZmMADS67 and ZmMADS69 are all targets of artificial selection and likely contributed to the spread of maize from the tropics to temperate zones[75, 76].

    In addition, a few genes regulating the photoperiod pathway and contributing to the acclimation of maize to higher latitudes in North America have been cloned, including Vgt1, ZmCCT (also named ZmCCT10), ZmCCT9 and ZmELF3.1. Vgt1 was shown to act as a cis-regulatory element of ZmRap2.7, and a MITE TE located ~70 kb upstream of Vgt1 was found to be significantly associated with flowering time and was a major target for selection during the expansion of maize to the temperate and high-latitude regions[7779]. ZmCCT is another major flowering-time QTL and it encodes a CCT-domain protein homologous to rice Ghd7[80]. Its causal variation is a 5122-bp CACTA-like TE inserted ~2.5 kb upstream of ZmCCT10[72, 81]. ZmCCT9 was identified a QTL for days to anthesis (qDTA9). A Harbinger-like TE located ~57 kb upstream of ZmCCT9 showed the most significant association with DTA and thus believed to be the causal variation[82]. Notably, the CATCA-like TE of ZmCCT10 and the Harbinger-like TE of ZmCCT9 are not observed in surveyed teosinte accessions, hinting that they are de novo mutations occurred after the initial domestication of maize[72, 82]. ZmELF3.1 was shown to underlie the flowering time QTL qFT3_218. It was demonstrated that ZmELF3.1 and its homolog ZmELF3.2 can form the maize Evening Complex (EC) through physically interacting with ZmELF4.1/ZmELF4.2, and ZmLUX1/ZmLUX2. Knockout mutants of Zmelf3.1 and Zmelf3.1/3.2 double mutant presented delayed flowering under both long-day and short-day conditions. It was further shown that the maize EC promote flowering through repressing the expression of several known flowering suppressor genes (e.g., ZmCCT9, ZmCCT10, ZmCOL3, ZmPRR37a and ZmPRR73), and consequently alleviating their inhibition on several maize florigen genes (ZCN8, ZCN7 and ZCN12). Insertion of two closely linked retrotransposon elements upstream of the ZmELF3.1 coding region increases the expression of ZmELF3.1, thus promoting flowering[83]. The increase frequencies of the causal TEs in Vgt1, ZmCCT10, ZmCCT9 and ZmELF3.1 in temperate maize compared to tropical maize highlight a critical role of these genes during the spread and adaptation of maize to higher latitudinal temperate regions through promoting flowering under long-day conditions[72,8183].

    In addition, Barnes et al.[84] recently showed that the High Phosphatidyl Choline 1 (HPC1) gene, which encodes a phospholipase A1 enzyme, contributed to the spread of the initially domesticated maize from the warm Mexican southwest to the highlands of Mexico and South America by modulating phosphatidylcholine levels. The Mexicana-derived allele harbors a polymorphism and impaired protein function, leading to accelerated flowering and better fitness in highlands.

    Besides the above characterized QTLs and genes, additional genetic elements likely also contributed to the pre-Columbia spreading of maize. Hufford et al.[85] proposed that incorporation of mexicana alleles into maize may helped the expansion of maize to the highlands of central Mexico based on detection of bi-directional gene flow between maize and Mexicana. This proposal was supported by a recent study showing evidence of introgression for over 10% of the maize genome from the mexicana genome[86]. Consistently, Calfee et al.[87] found that sequences of mexicana ancestry increases in high-elevation maize populations, supporting the notion that introgression from mexicana facilitating adaptation of maize to the highland environment. Moreover, a recent study examined the genome-wide genetic diversity of the Zea genus and showed that dozens of flowering-related genes (such as GI, BAS1 and PRR7) are associated with high-latitude adaptation[88]. These studies together demonstrate unequivocally that introgression of genes from Mexicana and selection of genes in the photoperiod pathway contributed to the spread of maize to the temperate regions.

    The so far cloned genes involved in pre-Columbia spread of maize are summarized in Fig. 2 and Table 2.

    Figure 2.  Genes involved in Pre-Columbia spread of maize to higher latitudes and the temperate regions. The production of world maize in 2020 is presented by the green bar in the map from Ritchie et al. (2023). Ritchie H, Rosado P, and Roser M. 2023. "Agricultural Production". Published online at OurWorldInData.org. Retrieved from: 'https:ourowrldindata.org/agricultural-production' [online Resource].
    Table 2.  Flowering time related genes contributing to Pre-Columbia spread of maize.
    GeneFunctional annotationCausative changeReferences
    ZCN8Florigen proteinSNP-1245 and Indel-2339 in promoter[73, 74]
    DLF1Basic leucine zipper transcription factorUnknown[75]
    ZmMADS69MADS-box transcription factorUnknown[76]
    ZmRap2.7AP2-like transcription factorMITE TE inserted ~70 kb upstream[7779]
    ZmCCTCCT-domain protein5122-bp CACTA-like TE inserted ~2.5 kb upstream[72,81]
    ZmCCT9CCT transcription factorA harbinger-like element at 57 kb upstream[82]
    ZmELF3.1Unknownwo retrotransposons in the promote[84]
    HPC1Phospholipase A1 enzymUnknown[83]
    ZmPRR7UnknownUnknown[88]
    ZmCOL9CO-like-transcription factorUnknown[88]
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    Subsequent to domestication ~9,000 years ago, maize has been continuously subject to human selection during the post-domestication breeding process. Through re-sequencing analysis of 35 improved maize lines, 23 traditional landraces and 17 wild relatives, Hufford et al.[15] identified 484 and 695 selective sweeps during maize domestication and improvement, respectively. Moreover, they found that about a quarter (23%) of domestication sweeps (107) were also selected during improvement, indicating that a substantial portion of the domestication loci underwent continuous selection during post-domestication breeding.

    Genetic improvement of maize culminated in the development of high planting density tolerant hybrid maize to increase grain yield per unit land area[89, 90]. To investigate the key morphological traits that have been selected during modern maize breeding, we recently conducted sequencing and phenotypic analyses of 350 elite maize inbred lines widely used in the US and China over the past few decades. We identified four convergently improved morphological traits related to adapting to increased planting density, i.e., reduced leaf angle, reduced tassel branch number (TBN), reduced relative plant height (EH/PH) and accelerated flowering. Genome-wide Association Study (GWAS) identified a total of 166 loci associated with the four selected traits, and found evidence of convergent increases in allele frequency at putatively favorable alleles for the identified loci. Moreover, genome scan using the cross-population composite likelihood ratio approach (XP-CLR) identified a total of 1,888 selective sweeps during modern maize breeding in the US and China. Gene ontology analysis of the 5,356 genes encompassed in the selective sweeps revealed enrichment of genes related to biosynthesis or signaling processes of auxin and other phytohormones, and in responses to light, biotic and abiotic stresses. This study provides a valuable resource for mining genes regulating morphological and physiological traits underlying adaptation to high-density planting[91].

    In another study, Li et al.[92] identified ZmPGP1 (ABCB1 or Br2) as a selected target gene during maize domestication and genetic improvement. ZmPGP1 is involved in auxin polar transport, and has been shown to have a pleiotropic effect on plant height, stalk diameter, leaf length, leaf angle, root development and yield. Sequence and phenotypic analyses of ZmPGP1 identified SNP1473 as the most significant variant for kernel length and ear grain weight and that the SNP1473T allele is selected during both the domestication and improvement processes. Moreover, the authors identified a rare allele of ZmPGP1 carrying a 241-bp deletion in the last exon, which results in significantly reduced plant height and ear height and increased stalk diameter and erected leaves, yet no negative effect on yield[93], highlighting a potential utility in breeding high-density tolerant maize cultivars.

    Shade avoidance syndrome (SAS) is a set of adaptive responses triggered when plants sense a reduction in the red to far-red light (R:FR) ratio under high planting density conditions, commonly manifested by increased plant height (and thus more prone to lodging), suppressed branching, accelerated flowering and reduced resistance to pathogens and pests[94, 95]. High-density planting could also cause extended anthesis-silking interval (ASI), reduced tassel size and smaller ear, and even barrenness[96, 97]. Thus, breeding of maize cultivars of attenuated SAS is a priority for adaptation to increased planting density.

    Extensive studies have been performed in Arabidopsis to dissect the regulatory mechanism of SAS and this topic has been recently extensively reviewed[98]. We recently showed that a major signaling mechanism regulating SAS in Arabidopsis is the phytochrome-PIFs module regulates the miR156-SPL module-mediated aging pathway[99]. We proposed that in maize there might be a similar phytochrome-PIFs-miR156-SPL regulatory pathway regulating SAS and that the maize SPL genes could be exploited as valuable targets for genetic improvement of plant architecture tailored for high-density planting[100].

    In support of this, it has been shown that the ZmphyBs (ZmphyB1 and ZmphyB2), ZmphyCs (ZmphyC1 and ZmphyC2) and ZmPIFs are involved in regulating SAS in maize[101103]. In addition, earlier studies have shown that as direct targets of miR156s, three homologous SPL transcription factors, UB2, UB3 and TSH4, regulate multiple agronomic traits including vegetative tillering, plant height, tassel branch number and kernel row number[44, 104]. Moreover, it has been shown that ZmphyBs[101, 105] and ZmPIF3.1[91], ZmPIF4.1[102] and TSH4[91] are selective targets during modern maize breeding (Table 3).

    Table 3.  Selective genes underpinning genetic improvement during modern maize breeding.
    GenePhenotypeFunctional annotationSelection typeCausative changeReferences
    ZmPIF3.1Plant heightBasic helix-loop-helix transcription factorIncreased expressionUnknown[91]
    TSH4Tassel branch numberTranscription factorAltered expressionUnknown[91]
    ZmPGP1Plant architectureATP binding cassette transporterAltered expressionA 241 bp deletion in the last exon of ZmPGP1[92, 93]
    PhyB2Light signalPhytochrome BAltered expressionA 10 bp deletion in the translation start site[101]
    ZmPIF4.1Light signalBasic helix-loop-helix transcription factorAltered expressionUnknown[102]
    ZmKOB1Grain yieldGlycotransferase-like proteinProtein functionUnknown[121]
     | Show Table
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    In a recent study to dissect the signaling process regulating inflorescence development in response to the shade signal, Kong et al.[106] compared the gene expression changes along the male and female inflorescence development under simulated shade treatments and normal light conditions, and identified a large set of genes that are co-regulated by developmental progression and simulated shade treatments. They found that these co-regulated genes are enriched in plant hormone signaling pathways and transcription factors. By network analyses, they found that UB2, UB3 and TSH4 act as a central regulatory node controlling maize inflorescence development in response to shade signal, and their loss-of-function mutants exhibit reduced sensitivity to simulated shade treatments. This study provides a valuable genetic source for mining and manipulating key shading-responsive genes for improved tassel and ear traits under high density planting conditions.

    Nowadays, global maize production is mostly provided by hybrid maize, which exhibits heterosis (or hybrid vigor) in yields and stress tolerance over open-pollinated varieties[3]. Hybrid maize breeding has gone through several stages, from the 'inbred-hybrid method' stage by Shull[107] and East[108] in the early twentieth century, to the 'double-cross hybrids' stage (1930s−1950s) by Jones[109], and then the 'single-cross hybrids' stage since the 1960s. Since its development, single-cross hybrid was quickly adopted globally due to its superior heterosis and easiness of production[3].

    Single-cross maize hybrids are produced from crossing two unrelated parental inbred lines (female × male) belonging to genetically distinct pools of germplasm, called heterotic groups. Heterotic groups allow better exploitation of heterosis, since inter-group hybrids display a higher level of heterosis than intra-group hybrids. A specific pair of female and male heterotic groups expressing pronounced heterosis is termed as a heterotic pattern[110, 111]. Initially, the parental lines were derived from a limited number of key founder inbred lines and empirically classified into different heterotic groups (such as SSS and NSS)[112]. Over time, they have expanded dramatically, accompanied by formation of new 'heterotic groups' (such as Iodent, PA and PB). Nowadays, Stiff Stalk Synthetics (SSS) and PA are generally used as FHGs (female heterotic groups), while Non Stiff Stalk (NSS), PB and Sipingtou (SPT) are generally used as the MHGs (male heterotic groups) in temperate hybrid maize breeding[113].

    With the development of molecular biology, various molecular markers, ranging from RFLPs, SSRs, and more recently high-density genome-wide SNP data have been utilized to assign newly developed inbred lines into various heterotic groups, and to guide crosses between heterotic pools to produce the most productive hybrids[114116]. Multiple studies with molecular markers have suggested that heterotic groups have diverged genetically over time for better heterosis[117120]. However, there has been a lack of a systematic assessment of the effect and contribution of breeding selection on phenotypic improvement and the underlying genomic changes of FHGs and MHGs for different heterotic patterns on a population scale during modern hybrid maize breeding.

    To systematically assess the phenotypic improvement and the underlying genomic changes of FHGs and MHGs during modern hybrid maize breeding, we recently conducted re-sequencing and phenotypic analyses of 21 agronomic traits for a panel of 1,604 modern elite maize lines[121]. Several interesting observations were made: (1) The MHGs experienced more intensive selection than the FMGs during the progression from era I (before the year 2000) to era II (after the year 2000). Significant changes were observed for 18 out of 21 traits in the MHGs, but only 10 of the 21 traits showed significant changes in the FHGs; (2) The MHGs and FHGs experienced both convergent and divergent selection towards different sets of agronomic traits. Both the MHGs and FHGs experienced a decrease in flowering time and an increase in yield and plant architecture related traits, but three traits potentially related to seed dehydration rate were selected in opposite direction in the MHGs and FHGs. GWAS analysis identified 4,329 genes associated with the 21 traits. Consistent with the observed convergent and divergent changes of different traits, we observed convergent increase for the frequencies of favorable alleles for the convergently selected traits in both the MHGs and FHGs, and anti-directional changes for the frequencies of favorable alleles for the oppositely selected traits. These observations highlight a critical contribution of accumulation of favorable alleles to agronomic trait improvement of the parental lines of both FHGs and MHGs during modern maize breeding.

    Moreover, FST statistics showed increased genetic differentiation between the respective MHGs and FHGs of the US_SS × US_NSS and PA × SPT heterotic patterns from era I to era II. Further, we detected significant positive correlations between the number of accumulated heterozygous superior alleles of the differentiated genes with increased grain yield per plant and better parent heterosis, supporting a role of the differentiated genes in promoting maize heterosis. Further, mutational and overexpressional studies demonstrated a role of ZmKOB1, which encodes a putative glycotransferase, in promoting grain yield[121]. While this study complemented earlier studies on maize domestication and variation maps in maize, a pitfall of this study is that variation is limited to SNP polymorphisms. Further exploitation of more variants (Indels, PAVs, CNVs etc.) in the historical maize panel will greatly deepen our understanding of the impact of artificial selection on the maize genome, and identify valuable new targets for genetic improvement of maize.

    The ever-increasing worldwide population and anticipated climate deterioration pose a great challenge to global food security and call for more effective and precise breeding methods for crops. To accommodate the projected population increase in the next 30 years, it is estimated that cereal production needs to increase at least 70% by 2050 (FAO). As a staple cereal crop, breeding of maize cultivars that are not only high-yielding and with superior quality, but also resilient to environmental stresses, is essential to meet this demand. The recent advances in genome sequencing, genotyping and phenotyping technologies, generation of multi-omics data (including genomic, phenomic, epigenomic, transcriptomic, proteomic, and metabolomic data), creation of novel superior alleles by genome editing, development of more efficient double haploid technologies, integrating with machine learning and artificial intelligence are ushering the transition of maize breeding from the Breeding 3.0 stage (biological breeding) into the Breeding 4.0 stage (intelligent breeding)[122, 123]. However, several major challenges remain to be effectively tackled before such a transition could be implemented. First, most agronomic traits of maize are controlled by numerous small-effect QTL and complex genotype-environment interactions (G × E). Thus, elucidating the contribution of the abundant genetic variation in the maize population to phenotypic plasticity remains a major challenge in the post-genomic era of maize genetics and breeding. Secondly, most maize cultivars cultivated nowadays are hybrids that exhibit superior heterosis than their parental lines. Hybrid maize breeding involves the development of elite inbred lines with high general combining ability (GCA) and specific combining ability (SCA) that allows maximal exploitation of heterosis. Despite much effort to dissect the mechanisms of maize heterosis, the molecular basis of maize heterosis is still a debated topic[124126]. Thirdly, only limited maize germplasm is amenable to genetic manipulation (genetic transformation, genome editing etc.), which significantly hinders the efficiency of genetic improvement. Development of efficient genotype-independent transformation procedure will greatly boost maize functional genomic research and breeding. Noteworthy, the Smart Corn System recently launched by Bayer is promised to revolutionize global corn production in the coming years. At the heart of the new system is short stature hybrid corn (~30%−40% shorter than traditional hybrids), which offers several advantages: sturdier stems and exceptional lodging resistance under higher planting densities (grow 20%−30% more plants per hectare), higher and more stable yield production per unit land area, easier management and application of plant protection products, better use of solar energy, water and other natural resources, and improved greenhouse gas footprint[127]. Indeed, a new age of maize green revolution is yet to come!

    This work was supported by grants from the Key Research and Development Program of Guangdong Province (2022B0202060005), National Natural Science Foundation of China (32130077) and Hainan Yazhou Bay Seed Lab (B21HJ8101). We thank Professors Hai Wang (China Agricultural University) and Jinshun Zhong (South China Agricultural University) for valuable comments and helpful discussion on the manuscript. We apologize to authors whose excellent work could not be cited due to space limitations.

  • The authors declare that they have no conflict of interest. Haiyang Wang is an Editorial Board member of Seed Biology who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and his research groups.

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  • Cite this article

    Liu H, Hao L, Zhang X, Zhang Y, Wang H, et al. 2024. Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume. Tropical Plants 3: e010 doi: 10.48130/tp-0024-0010
    Liu H, Hao L, Zhang X, Zhang Y, Wang H, et al. 2024. Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume. Tropical Plants 3: e010 doi: 10.48130/tp-0024-0010

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Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume

Tropical Plants  3 Article number: e010  (2024)  |  Cite this article

Abstract: The CBL gene family is an important family in the Ca2+ mediated signal transduction pathway in plants and plays a crucial role in plant stress responses and growth development. However, research on the response of members of the Prunus mume CBL gene family to low temperature stress remains scarce. In this study, we systematically analyzed the protein physicochemical properties, chromosome localization, phylogenetic evolution, gene structure, conserved domains, cis-acting elements, and gene expression patterns in response to low temperature stress of members of the P. mume CBL gene family using bioinformatics tools. Six PmCBL gene family members were identified in the P. mume genome. Phylogenetic trees were constructed, revealing three subfamilies named Group I, Group II, and Group III. In the P. mume gene family, PmCBL4 and PmCBL5 were paralogous genes. The members of the P. mume CBL gene family were unevenly distributed on three chromosomes. The CBL encoding protein, the number of isoelectric points (pI), the number of introns and exons of the six gene families were different. Analysis of the upstream 700 bp promoter sequences of the P. mume CBL gene family revealed the presence of various types of cis-acting elements involved in non-biological stress responses. Among the six identified genes, each gene exhibited different expression patterns in response to low temperature. Among them, the up-regulated expression of PmCBL5 was the largest, and the expression of PmCBL1, PmCBL3 and PmCBL5 showed the up-regulated trend. These results indicated that PmCBL1, PmCBL3, PmCBL5, and PmCBL6 were key genes involved in the response of P. mume to low temperature stress. This study provided comprehensive and systematic analysis of the P. mume CBL gene family members and identified key genes involved in the response to low temperature stress, thereby providing genetic resources for molecular breeding programs aimed at enhancing cold resistance in P. mume.

    • In plants, Ca2+ is an integral element in several stress related signallings. During environmental stresses, the cytosolic Ca2+ concentration tends to rise rapidly, which is further co-ordinate with EF-hand domain-containing proteins[1]. Low temperature stress is an environmental challenge that severely limits the geographical distribution and survival rate of certain perennial plants[2]. Nowadays, some plants have evolved specific and effective molecular mechanisms to resist cold damage, and many functional genes involved in cold response have been identified in plants, with some of these genes being closely associated with Ca2+. Stomatal immunity is regulated by pathogen-associated molecular patterns (PAMPs)- and abscisic acid (ABA)-triggered signalling in different ways. Cytoplasmic Ca2+ signature in the guard cells plays a vital function in stomatal immunity, but the mechanism of Ca2+ import is unknown. In the study of the mechanism of PAMPs triggering stomatal closure, it was found that four types of CNGCs (CNGC2, 5, 6, 9, and 12) were enriched in Arabidopsis thaliana guard cells and involved in ABA induced cytoplasmic Ca2+ oscillations. However, only some inactive stomatal movements indicate that other Ca2+ channels (possibly OSCAs) are actively involved in Ca2+ mediated stomatal immunity. In addition, CDPKs mediated phosphorylation of CNGC6 also indicates the existence of alternative pathways. However, the interrelationships between various Ca2+ channels and their mechanisms of simultaneous activation during stomatal defense need to be validated in future research. In addition, ROS induced Ca2+ signaling is not affected in cells, indicating the function of these channels downstream of the ROS pathway[3]. Ca2+ signaling represents a universal transduction signal in plants, mediated by a complex network of Ca2+ binding proteins, many of which function as Ca2+ sensors and act on downstream responses[4]. Calcium-dependent protein kinases (CDPKs) form functional complexes with CBL-interacting protein kinases (CIPKs), enabling plants to respond to various environmental signals and regulate ion fluxes[5]. The CBL-CIPK complex plays a crucial role in signal transduction pathways, where Ca2+ serves as a second messenger, particularly in the regulation of ion transporter activities in response to non-biological signals[6]. In poplar trees, the PeCBL/PeCIPK complex has been identified and shown to play a role in Na+/K+ homeostasis[7].

      Over the past few decades, numerous CBL-CIPK complexes have been shown to be involved in signal transduction under non-biological stress. The CBL-CIPK signaling network system, composed of CBLs and their target proteins CIPKs (CBL-interacting protein kinases), plays a significant role in plant responses to various abiotic stresses such as drought, salinity, and low temperature[811]. The plant CBL gene family typically consists of 10 members and was initially discovered in A. thaliana, but has since been successfully isolated from various crops such as Zea mays, Populus alba, Gossypium sp, and Oryza sativa[1114]. Numerous studies have shown that calcineurin B like proteins (CBL) play an important role in plant stress response. Recent studies on Setaria italica have found that SiCBL3 is widely involved in the response of S. italica seedlings to various abiotic stress conditions, such as PEG, salt, high temperature, low temperature, and ABA; SiCBL3 is highly expressed during normal heading and filling stages, and is extensively induced under drought stress during the jointing, heading, and filling stages of S. italica[15]. Studying the gene structure, distribution, and expression characteristics of CBL genes in plant genomes is of great significance in gaining a deeper understanding of their roles in growth, development, and stress responses. The members of the CBL gene family show a strong conservation in their structural features, with most CBL family members containing 7~8 introns, and the positions of these introns within the coding region are relatively fixed[14,16]. The differences in domain structures among different CBL proteins may contribute to their varying abilities to bind calcium ions, providing a foundation for CBLs to sense changes in Ca2+ concentration triggered by various stimulus signals[17]. In recent years, CBL genes from multiple species have been successfully isolated and functionally validated[1824]. In A. thaliana, AtCBL1 and AtCBL9 are localized to the plasma membrane, and both genes show expression throughout development, although their expression levels are relatively weak in roots. AtCBL5 is also a calcium signaling component located on the plasma membrane, and it exhibits high-level expression in leaves[18,19]. Numerous studies have demonstrated that CBLs exhibit different expression patterns in response to various stressors and during growth and development processes. For example, AtCBL1 is strongly induced by low temperature but is not influenced by exogenous ABA stress[18,20], while AtCBL9 is induced by ABA stress[19]. Although significant progress has been made in the study of CBL genes, most research has been limited to a few plant species, and the majority of CBL genes remain unisolated and functionally characterized.

      P. mume is an important ornamental woody plant. It is native to the Sichuan-Yunnan-Tibet region and mainly distributed in the Yangtze River Basin (China). To date, research on freezing resistance in P. mume has primarily focused on the ICE-CBF signaling pathway, while studies on the upstream Ca2+ signaling pathway have been relatively limited. Building upon previous studies on the response of CIPK gene family to low temperature stress, this research focused on identifying and investigating the response to low temperature stress of CBL protein family members that interact with CIPK proteins. The use of bioinformatics analysis methods, that is, based on existing bioinformation databases and resources, the use of mature bioinformatics tools to solve bioinformatics problems. In this study, all CBL gene family members were identified based on the whole genome sequence information of P. mume. The number of CBL genes, gene structure, evolution, and expression patterns under low temperature stress were analyzed at the genome level, providing insights into the biological functions of these family members. Additionally, this study provides an important theoretical basis for the breeding of cold resistant P. mume varieties.

    • The P. mume cultivar 'Zao Lve' was used as a plant material. The 'Zao Lve' variety, after years of cold domestication, could survive and bloom when grown in open fields in North China. For the experiment, two-year-old grafted seedlings of the 'Zao Lve' variety were selected and subjected to low-temperature treatment.

    • The data for the P. mume gene family in this study were obtained from the P. mume database, including the P. mume gff annotation file and P. mume CDS encoding protein sequences. The CBL gene family A. thaliana was derived from the Uniprot gene database (www.uniprot.org)[25]. The CBL domain files for each gene member of the obtained A. thaliana gene family were determined and downloaded using PFAM (https://precisionflange.com/)[26]. To identify the members of the P. mume CBL gene family, the ten published A. thaliana CBL gene family members were used as query genes. The hmmsearch plugin in the hmmer software was utilized for the search (e-value set at 10−5, with other parameters set to default values) to screen for CBL homologous sequences from the P. mume gene family members. The results were then searched against the A. thaliana family in the String database, and members of the P. mume CBL gene family with a similarity greater than 50% were selected.

    • Based on the identified sequence IDs, the GXF Select tool in TBtools was used to extract the annotation information of the identified sequences. This allowed us to obtain the chromosome numbers and location information of the P. mume CBL gene family members. The six CBL family members were then named based on their respective location information. The ExPASy-Protparam online tool (https://web.expasy.org/protparam) was used to analyze the physicochemical properties of the gene family members. It involved amino acid count statistics and predicted protein properties such as molecular weight (MW), theoretical isoelectric point (pI), aliphatic index, instability index, and grand average of hydropathicity (GRAVY). The SignalP v6.0 online tool was used to predict signal peptides, while the TMHMM v2.0 online tool was utilized to predict transmembrane helices. The Cell-PLoc v2.0 online tool (www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2) was employed for subcellular localization prediction.

    • In this study, the phylogenetic tree of P. mume CBL gene family was analyzed by Neighbor-Joining Algorithm method[27]. The MEGA 7.0 (7170509-X86_64) software was used for phylogenetic analysis of the gene family. The protein sequences of CBLs from A. thaliana, O. sativa, Vitis vinifera and Nicotiana tabacum were obtained from the Uniprot database (www.uniprot.org)[25]. The MUSCLE program in the software was used for multiple sequence alignment of the selected CBL protein sequences with their corresponding A. thaliana homologous protein sequences (using default parameters). The aligned results were subjected to phylogenetic analysis using the Neighbor-Joining (NJ) method, with the number of differences as the computational model and pairwise deletion as the gap handling option. Bootstrap support was set to 500 for tree reliability assessment. The resulting phylogenetic tree was visualized and beautified using the Evolview online tool (www.evolgenius.info/evolview/#/)[28].

    • The identified CBL genes were assigned to their corresponding positions on the P. mume genome. The location information of each gene on the chromosomes was obtained using the online tool MG2C (http://mg2c.iask.in/mg2c_v2.1/) to generate a chromosomal localization map.

    • Multiple sequence alignment of the selected CBL protein sequences was performed using the Clustal W program in MEGA software (with default parameters). The MEME online tool (https://meme-suite.org/meme/) was utilized for motif analysis to predict conserved motifs and domains present in the protein sequences. Based on the identified P. mume CBL gene family with six gene members, the Gene Structure View (Advanced) tool in TBtools software was used along with the downloaded P. mume annotation information gff file from the P. mume database to analyze the gene structure of the P. mume CBL gene family.

    • In order to analyze the potential cis-acting elements in the promoter sequences of the P. mume CBL gene family, the upstream 700 bp promoter sequence regions were downloaded in bulk from the ATG start codon. The obtained promoter sequences were then analyzed for cis-acting elements using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html)[29]. The resulting Tab file was opened in an Excel spreadsheet, and the desired cis-acting elements were filtered. Finally, the Simple BioSequence Viewer function in TBtools software was used to visualize and plot the identified cis-acting elements.

    • In order to further investigate the expression patterns of PmCBL genes, We collected transcriptomic expression profile data from different tissues (roots, stems, leaves, buds, and fruits) and during the natural overwintering process (November, December, January, and February) in P. mume[30].

    • Seedlings cultivated under long-day conditions (16 h light/8 h dark) at 24 °C were used to investigate the effect of PmCBL genes on cold response. The seedlings were incubated at 4 °C, mimicking a cold environment. One-year-old branches that underwent 4 °C treatment were selected for sampling at various time points including 0, 1, 4, 6, 12, and 24 h. Total RNA was extracted from the samples using a suitable method. First-strand cDNA synthesis was performed using the TIANScript First Strand cDNA Synthesis Kit (Tiangen, China). qRT-PCR was performed using the PikoReal Real-Time PCR System (Thermo Fisher Scientific, CA, USA) and SYBR Premix ExTaq TM (TaKaRa, Dalian, China). The reaction was carried out in a 10 μL system, which included 5 μL of SYBR Premix ExTaqII, 0.25 μL each of forward and reverse primers, 0.5 μL of cDNA, and 3 μL of ddH2O. The PCR program consisted of 40 cycles at 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 40 s, followed by a final extension at 60 °C for 30 s. Three replicates were performed for each sample. The internal reference gene used was PP2A from P. mume. The relative expression levels of PmCBL genes were calculated using the 2−ΔΔCᴛ method. Statistical analysis with standard deviation was performed on the final data.

    • Through bioinformatics analysis methods, a total of six CBL gene family members were identified (Fig. 1a) and named as PmCBL1~PmCBL6 (Table 1). These six CBL genes were unevenly distributed on the chromosomes. PmCBL1 was located on chromosome 1, PmCBL2 and PmCBL3 were located on chromosome 5, and PmCBL4, PmCBL5, and PmCBL6 were located on chromosome 7. In the PmCBL family, the coding sequence (CDS) of PmCBL6 was the longest, comprising 2,475 bp. PmCBL1 had the second-longest CDS with 2,418 bp, followed by PmCBL3 with 1,893 bp, PmCBL2 with 1,805 bp, PmCBL5 with 1,564 bp, and PmCBL4 with the shortest CDS of 1,252 bp. The subcellular localization prediction of these six genes was all on the cell membrane. Through analysis of conserved protein domains, it was found that PmCBLs contain six conserved motifs (Motif 1~6) (Fig. 1b). Specifically, PmCBL2, PmCBL3, PmCBL5, and PmCBL6 contained all six motifs (Motif 1~6), while PmCBL1 and PmCBL4 contained Motif 1~4 and Motif 6 but did not contain Motif 5. From the results of protein multiple sequence alignment among P. mume, A. thaliana, V. vinifera, and O. sativa, it could be observed that P. mume showed higher homology with A. thaliana and V. vinifera, while the homology with O. sativa was relatively lower (Fig. 2).

      Figure 1. 

      Phylogenetic analysis, protein motif structure, chromosome localization, and gene structure analysis of PmCBL genes. (a) Phylogenetic analysis of PmCBL genes. (b) Protein motif structure of PmCBL genes. (c) Chromosome localization analysis of PmCBL genes. (d) Gene structure analysis of PmCBL genes.

      Table 1.  Members of the P. mume PmCBL gene family and their main molecular characteristics and information.

      Gene nameGene IDChromosomePositionSubcelllar localizationCDS (bp)Intron
      PmCBL1Pm000366Chr012287536~2289774Cell membrane2,4185
      PmCBL2Pm018367Chr0517870110~17871915Cell membrane1,8054
      PmCBL3Pm019754Chr0525555186~25557079Cell membrane1,8935
      PmCBL4Pm024130Chr0710584318~10585570Cell membrane1,2524
      PmCBL5Pm024134Chr0710621981~10623545Cell membrane1,5645
      PmCBL6Pm024135Chr0710622516~10628186Cell membrane2,4745

      Figure 2. 

      Protein amino acid sequence comparison diagram. (a) Protein amino acid sequence comparison diagram between P. mume and A. thaliana. (b) Protein amino acid sequence comparison diagram between P. mume and V. vinifera. (c) Protein amino acid sequence comparison diagram between P. mume and O. sativa.

    • The physicochemical properties of the six CBL homologous sequences were analyzed (Table 2). The results showed that the amino acid (aa) numbers of the PmCBL protein sequences ranged from 186 (PmCBL4) to 226 (PmCBL1), with molecular weights (MV) ranging from 21.66 kDa (PmCBL4) to 26.05 kDa (PmCBL1). The theoretical isoelectric points (pI) ranged from 4.57 to 5.14. The instability index values ranged from 35.97 to 49.06, with only PmCBL1 having an instability index below 40, indicating high stability of CBL protein. The grand average of hydropathicity (GRAVY) values for PmCBL1 ~ PmCBL6 were all negative, indicating that these six proteins were hydrophilic. The aliphatic index values ranged from 90.09~96.96, indicating that all six proteins were lipophilic. None of the six gene family members contained signal peptides or transmembrane helices.

      Table 2.  Physicochemical properties of P. mume CBL gene family members.

      Protein nameGene IDNumber of amino acidsMolecular weightThereotical pIInstability indexSignal peptide
      PmCBL1Pm00036622626052.714.8249.06NO
      PmCBL2Pm01836721724903.525.1435.97NO
      PmCBL3Pm01975421224429.824.7346.65NO
      PmCBL4Pm02413018621665.754.640.14NO
      PmCBL5Pm02413421324720.174.5740.98NO
      PmCBL6Pm02413521825144.714.8646.47NO
    • To understand the evolutionary relationship between members of the P. mume CBL gene family, a phylogenetic tree was constructed using the selected six PmCBL genes, 17 OsCBLs genes from O. sativa, 16 VvCBLs genes from V. vinifera, 20 NtCBLs genes from N. tabacum and 10 AtCBLs genes from the model plant A. thaliana (Fig. 3). The results of the evolutionary analysis showed that the CBL gene family could be divided into three groups, each containing among them, P. mume Group I included PmCBL2, PmCBL3, PmCBL4, PmCBL5, and PmCBL6. Group III consisted of PmCBL1. In Group I, PmCBL4 and PmCBL5 were paralogous gene pairs. This suggested that the CBL gene in P. mume underwent expansion, expansion and replication. Some genes in AtCBLs, NtCBLs, VvCBLs and PmCBLs could be considered as orthologous gene pairs, such as AtCBL4 and PmCBL3, NtCBL14 and PmCBL2, VvCBL8 and PmCBL6. The discovery of orthologous gene pairs suggests the existence of ancient CBL genes before P. mume and A. thaliana, N. tabacum, V. vinifera were also similar.

      Figure 3. 

      Phylogenetic tree of P. mume, A. thaliana, O. sativa, N. tabacum and V. vinifera. The green checkmark represents V. vinifera, the black square represents O. sativa, the blue circle represents A. thaliana, the red star represents N. tabacum, and the purple triangle represents P. mume.

    • Further analysis of the gene structure of the P. mume CBL gene family revealed that the six PmCBL genes share a similar overall gene structure (Fig. 1d). Specifically, PmCBL3 had eight exons separated by introns. PmCBL4 had seven exons with approximately equal sizes separated by introns. PmCBL2 had six exons separated by introns, with two exons being relatively far apart. PmCBL5 had eight exons separated by introns, and the sizes of these eight exons were roughly similar with a similar distance between each pair of exons. PmCBL6 had eight exons separated by introns, with some exons having larger inter-exon distances. PmCBL1 also had eight exons separated by introns, and similarly, some exons had larger inter-exon distances.

      Based on the results from the MG2C online tool (Fig. 1c), the six identified P. mume CBL genes were found to be located on three chromosomes. PmCBL2 and PmCBL3 were located on one chromosome, PmCBL4, PmCBL5, and PmCBL6 were located on another chromosome, and PmCBL1 was located on a separate chromosome.

    • In order to further investigate the potential functional roles of PmCBL genes, the 700 bp sequence upstream of the start codon of each PmCBL gene was extracted as its promoter region. Cis-acting element analysis was performed on this region, focusing on important elements that had been extensively studied and were associated with plant growth and development, as well as stress responses. (Table 3, Fig. 4) The results showed that a total of 17 cis-acting element types responsive to plant hormones and stress were identified, and there were differences in the types and quantities of elements among different genes. Among them, all genes contained common cis-acting elements (CAAT-box) in their promoter and enhancer regions, with slight differences in the number of elements ranging from 2~6. PmCBL3 had the highest number of six (CAAT-box elements) , while PmCBL4 had the lowest two. Two cis-acting regulatory elements involved in MeJA response (CGTCA-motif and TGACG-motif) were found, and five genes contained both of these elements, with an equal number in each gene. Five genes contained the core promoter element (TATA-box) located around the transcription start site −30, namely PmCBL1 ~ PmCBL5, but there was a large variation in the number of elements among the members, ranging from 4~11. PmCBL1 had one light-responsive element (GT1-motif), while the other genes did not contain it. There were four light-responsive elements identified: PmCBL1, PmCBL4, and PmCBL5 each contained one GATA-motif, PmCBL2 contained one I-box, PmCBL1 contained one TCCC-motif, and PmCBL3 contained one LAMP-element. Two cis-acting regulatory elements involved in zein protein metabolism regulation were found: O2-site and MBS. PmCBL3, PmCBL4, and PmCBL5 each contained one O2-site, PmCBL6 contained two O2-sites, and PmCBL4 and PmCBL6 each contained one MBS. One cis-acting regulatory element involved in light response (G-box) was identified, with PmCBL4 and PmCBL5 each containing one. One cis-acting regulatory element required for anaerobic induction ARE was found, with PmCBL2 and PmCBL5 each containing one. One cis-acting regulatory element (A-box) involved in cis-element regulation was found, with PmCBL5 and PmCBL6 each containing one. One cis-acting element involved in abscisic acid response ABRE was found, with PmCBL4 and PmCBL5 each containing one. One cis-acting element involved in auxin response (TGA-element) was found in PmCBL6. One cis-acting element involved in salicylic acid response (TCA-element) was found in PmCBL4.

      Table 3.  Analysis of cis-acting elements in the P. mume CBL gene family members.

      GenePmCBL1PmCBL2PmCBL3PmCBL4PmCBL5PmCBL6
      Gene IDPm000366Pm018367Pm019754Pm024130Pm024134Pm024135
      CAAT-box346244
      CGTCA-motif21121
      TGACG-motif21121
      TATA-box444118
      GT1-motif1
      TCCC-motif1
      GATA-motif111
      ARE11
      G-box11
      MBS11
      TGA-element1
      A-box11
      I-box1
      O2-site1112
      LAMP-element1
      ABRE11
      TCA-element1
      CAAT-box was a common cis-acting element in the promoter and enhancer regions. CGTCA-motif/TGACG-motif was cis-acting regulatory elements involved in MeJA response. TATA-box was a core promoter element located around the transcription start site (-30). GT1-motif was a light-responsive element. TCA-element was a cis-acting element involved in salicylic acid response. TGA-element was an element involved in auxin response. ABRE was a cis-acting element involved in abscisic acid response. A-box was a cis-acting regulatory element. ARE was a cis-acting element required for anaerobic induction. G-box was a cis-acting element involved in light response. O2-site/MBS was cis-acting regulatory elements involved in zein protein metabolism regulation. TCCC-motif/GATA-motif/I-box/LAMP-element was part of light-responsive elements.

      Figure 4. 

      Analysis of cis-acting elements in PmCBL genes.

    • By analyzing transcriptome data from different parts of the P. mume, tissue-specific expression patterns were observed in different members of the P. mume CBL gene family (Fig. 5). PmCBL1 showed higher expression levels in flower buds, PmCBL2 exhibited higher expression levels in roots, PmCBL3 and PmCBL6 had higher expression levels in leaves, and PmCBL4 and PmCBL5 showed higher expression levels in stems. From this, it could be observed that PmCBL3 and PmCBL6 had similar expression patterns, while PmCBL4 and PmCBL5 had similar expression patterns. This suggested that they belonged to the same Group I subfamily.

      Figure 5. 

      Expression patterns of PmCBL genes in different tissue parts of P. mume.

      By analyzing the transcriptome data of P. mume during wintering, the wintering process of P. mume could be divided into three stages: the early stages of overwintering, which was November (DD); midwinter, which was December (NDD); late overwintering which was January (LD); and naturalness, which was February (NF). The expression patterns of the P. mume CBL gene family could be obtained (see Fig. 6), with different genes showing different expression patterns. PmCBL1 showed an upregulation trend in all three stages compared to the NF stage, reaching its peak in the LD stage, with expression levels 1.23 times higher than the NF stage. PmCBL2 exhibited both upregulation and downregulation trends in the three stages compared to the NF stage. It reached its maximum expression level in the NDD stage, being 1.22 times higher than the NF stage. However, it reached its minimum expression level in the LD stage, showing a downregulation of 1.3 times compared to the NF stage. Overall, PmCBL2 displayed a downregulation trend. PmCBL3 showed an upregulation trend in all three stages compared to the NF stage, with the highest expression level observed in the DD stage, being 1.06 times higher than the NF stage. PmCBL4 showed a downregulation trend in all three stages compared to the NF stage, with the minimum expression level observed in the DD stage, showing a downregulation of 1.20 times compared to the NF stage. PmCBL5 exhibited a downregulation trend relative to the NF stage, reaching its minimum expression level in the NDD stage, showing a downregulation of 8.28 times compared to the NF stage. PmCBL6 showed an upregulation trend relative to the NF stage, with an upregulation of 1.42 times compared to the NF stage. PmCBL4 and PmCBL5 were known to had significant roles in the wintering process of P. mume.

      Figure 6. 

      The expression pattern of PmCBLs genes during overwintering. DD, November; NDD, December; LD, January; NF, February.

    • To further investigate the expression patterns of PmCBL genes in response to low temperature stress, qRT-PCR experiments were conducted to examine the expression levels of PmCBLs under cold treatment. During the treatment at 4 °C, the expression levels of PmCBL genes showed an upregulation or downregulation trend over a 24-h time course (Fig. 7). PmCBL1, PmCBL2, PmCBL3, PmCBL5, and PmCBL6 showed an upregulation trend in their expression levels. Among them, PmCBL1 and PmCBL5 exhibited the highest expression levels at 24 h, being 12.18-fold and 50.11-fold higher than the pre-treatment levels, respectively. PmCBL2 and PmCBL6 reached their maximum expression levels at 6 h, with a 5.50-fold and 8.90-fold increase, respectively, compared to the pre-treatment levels. PmCBL3 showed the highest expression level at 12 h, which was 11.22-fold higher than the pre-treatment level. PmCBL4 showed a downregulation trend compared to the pre-treatment levels.

      Figure 7. 

      Expression levels of PmCBL genes under 4 °C treatment.

    • Currently, members of the CBL gene family have been identified in various vegetables, fruits, and cereal crops. The CBL gene family has been extensively studied in various plant species. In V. vinifera, a total of eight members of the CBL gene family were found[31]. Similarly, in O. sativa, 10 members of the CBL gene family were identified[32]. A. thaliana, known for its significance in plant research, has also revealed the presence of 10 CBL gene family members[11]. Solanum lycopersicum, commonly known as tomato, exhibited 13 characterized members of the CBL gene family[33]. Remarkably, Triticum aestivum, or wheat, stands out with the largest number of identified CBL gene family members, totaling 68[34]. Additionally, CBL gene family members have also been identified in Algae, Racomitrium canescens, Pteridophyta, and gourd plants. In this study, a bioinformatics approach was employed to obtain the structural domain files of the CBL gene family and perform an HMMER SEARCH. Through Pfam database, a total of six P. mume CBL gene family members were obtained. These findings shed light on the diversity and complexity of the CBL gene family across different plant species.

      The composition of introns and exons can reflect the evolutionary relationships within a gene family. It has been found that CBL genes in dicot model plants such as A. thaliana and poplar contained 6~7 introns. In the six identified P. mume CBL gene family members, all of them contained introns, with a ranged of 7~8 introns and minimal variation in the numbers. This suggested that introns in P. mume CBL genes might be more active during the evolutionary process compared to plants like A. thaliana and poplar. Generally, early-stage plant evolution tends to exhibit a higher enrichment of introns compared to later stages[35], with a higher rate of intron loss than gain[36]. To further explore the evolutionary relationships, phylogenetic trees were constructed using CBL proteins from A. thaliana, V. vinifera, O. sativa, N. tabacum and P. mume. The CBL proteins from these five species can be divided into three subfamilies, which may have evolved from different ancestral sequences. The six CBL genes of P. mume were distributed in Group I and Group III. In Group I, homologous gene pairs of P. mume and homologous gene pairs of P. mume and other species had been found. The identification of homologous gene pairs not only provides insights into the duplication and diversification processes within P. mume's own genome but also sheds light on the phylogenetic relationships between P. mume and other species. This discovery highlights the intricate mechanisms underlying the preservation and replication of genetic information, as well as the evolutionary connections that exist among different organisms.

      Low temperature conditions, also known as cold stress, pose a significant threat to plant growth. Freezing stress impedes the growth of most plants and poses a great risk to the cultivation of many perennial woody plants. Previous studies have found that T. aestivuml grown under normal temperature conditions are killed at freezing temperatures of approximately −5 °C. However, if the species undergoes cold acclimation, it can survive at temperatures as low as −20 °C[37]. In previous research on Pyrus, it is found that under low temperature (4 °C) stress, the expression levels of PbCBL2, PbCBL4, and PbCBL8 were upregulated, PbCBL1 and PbCBL3 exhibited downregulation in expression under low-temperature stress[38]. Additionally, in previous studies on V. vinifera, except for VvCBL5, which shows a significant downregulation expression trend under low temperature stress, all seven VvCBL genes show a significant upregulation expression trend[39]. In this study, the P. mume gene database was utilized to obtain the sequences of CBL gene family members. These sequences were used as probes for expression analysis, enabling the investigation of expression characteristics. The results from real-time fluorescence quantitative PCR demonstrated that the expression patterns of the six P. mume PmCBLs varied under low-temperature stress. This variation may be attributed to the involvement of PmCBLs in regulating the signaling pathways associated with low-temperature responses. During the 4 °C treatment, the expression levels of PmCBL genes showed an upregulation or downregulation trend over a 24 h time period. PmCBL1, PmCBL2, PmCBL3, PmCBL5, and PmCBL6 exhibited upregulation in expression, among which PmCBL1, PmCBL3, PmCBL5 and PmCBL6 showed the most significant upregulation, suggesting their crucial roles in regulating P. mume response to low-temperature stress. Through comparative analysis of P. mume, V. vinifera, and Pyrus, it can be seen that they have certain similarities in response to low temperature stress.

      Cold-responsive genes were cloned, and functional analysis was performed using the whole genome. The exact role of the c-repeat/DRE binding factor (CBF/DRE) in cold tolerance was studied in P. mume[25,40]. After cold treatment, the expression levels of PmCBLs showed either upregulation or downregulation, but these genes exhibited differential expression levels as shown in Fig. 6. These expression patterns, similar to their homologs, suggest that PmCBLs might play important roles in cold response. To date, numerous studies have demonstrated the important role of plant CBL genes in plant stress responses. For instance, AtCBL1 in A. thaliana can be strongly induced by non-biological stresses such as low temperature and injury but is not influenced by exogenous ABA[18,20]. On the other hand, AtCBL9 plays a role in both the ABA signaling pathway and ABA biosynthesis pathway and is primarily involved in the stress response of A. thaliana during the seedling stage[19]. ZmCBL4 in maize can significantly enhance salt tolerance in transgenic A. thaliana[13]. These findings highlight the significance of CBL genes in mediating plant responses to various stressors. Due to the signaling crosstalk among different stresses, multiple Ca2+ signals can be generated even under the same stress conditions. In addition, different CIPK target proteins may bind to the same sensor, and there may be functional redundancy between different CBL genes, making the entire CBL-CIPK signaling pathway complex and diverse. In order to gain a deeper understanding of the CBL-CIPK signaling pathway and how PmCBLs interacts with target proteins to activate downstream responses in response to low temperature stress, it is necessary to further explore other CBL genes.

      Through transcriptomic data analysis, the expression patterns of CBL proteins can be obtained. Previous studies have shown that N. tabacum CBL proteins exhibit tissue-specific expression patterns. NtCBL13 and NtCBL14 share similar expression patterns, with low expression levels in all tissues except for roots where they are expressed. On the other hand, NtCBL6, NtCBL8, NtCBL7, NtCBL4, NtCBL5, NtCBL1, and NtCBL9 exhibit similar expression patterns with high expression levels in various tissues, except NtCBL9 which shows lower expression levels in mature roots. The remaining NtCBL proteins have higher expression levels in flower tissues. Overall, NtCBL proteins are abundantly expressed in flowers, leaves, and roots[41]. According to the research conducted on P. mume, the gene expression of PmCBLs varies in different tissue parts of P. mume (Fig. 6). PmCBL1 showed higher expression levels in flower buds, PmCBL2 exhibited higher expression levels in roots, PmCBL3 and PmCBL6 showed higher expression levels in leaves, and PmCBL4 and PmCBL5 display higher expression levels in stems. The gene expression pattern analysis revealed that the expression levels of PmCBL genes were relatively high in stems. The differential expression levels of genes in different tissue parts might be a result of biological evolution. For N. tabacum, NtCBL6, NtCBL8, NtCBL7, NtCBL4, NtCBL5, NtCBL1, and NtCBL9 share similar expression patterns and belong to the Group A subfamily of the evolutionary system, while the remaining NtCBL proteins belong to the Group B subfamily[41]. As for P. mume, PmCBL3, and PmCBL6 exhibit similar expression patterns, and PmCBL4 and PmCBL5 also showed similar expression patterns, indicating that they belong to the Group I subfamily. Therefore, it can be observed that higher homology between genes leads to more similar gene expression patterns.

    • To summarize, we conducted a genome-wide identification of the P. mume CBL gene family for the first time. We identified six CBL genes, among which PmCBL1, PmCBL3, PmCBL5, and PmCBL6 showed the most significant upregulation, suggesting their crucial roles in regulating P. mume response to low-temperature stress. Therefore, this study indicated that PmCBLs might play a key role in enhancing freezing tolerance and winter hardiness in P. mume by modulating responses to low-temperature stress. This study contributed valuable genetic resources that could be utilized for the molecular breeding of cold-resistant P. mume.

    • The authors confirm contribution to the paper as follows: study conception and design: Li P, Zhang Q; data collection and analysis platform: Liu H, Hao L; data analysis and draft manuscript preparation: Liu H; provided help with the experiments: Zhang X, Zhang Y; manuscript revision: Wang H, Wang J, Liu Z, Zhang Q, Li P. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article.

      • The research was supported by the Science and Technology Project of Hebei Education Department under Grant No. BJK2024033, the Natural Science Foundation of Hebei Province under Grant No. C2021204184 and Hebei Agricultural University College Student Innovation and Entrepreneurship Training Program under Grant No. 202310086003.

      • The authors declare that they have no conflict of interest.

      • Received 22 October 2023; Accepted 27 February 2024; Published online 3 April 2024

      • For the first time, six PmCBL genes had been comprehensively and systematically identified in P. mume.

        The PmCBL gene family responded to cold stress, and PmCBL1/3/5/6 were identified as potential key genes involved in regulating cold tolerance in P. mume.

      • # Authors contributed equally: Haolin Liu, Lihong Hao

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of Hainan University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (7)  Table (3) References (41)
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    Liu H, Hao L, Zhang X, Zhang Y, Wang H, et al. 2024. Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume. Tropical Plants 3: e010 doi: 10.48130/tp-0024-0010
    Liu H, Hao L, Zhang X, Zhang Y, Wang H, et al. 2024. Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume. Tropical Plants 3: e010 doi: 10.48130/tp-0024-0010

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