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Article

The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response

by
Xiangshu Dong
1,†,
Jing Gao
1,†,
Meng Jiang
1,
Yuan Tao
1,
Xingbo Chen
1,
Xiaoshuang Yang
1,
Linglin Wang
1,
Dandan Jiang
1,
Ziwei Xiao
2,
Xuehui Bai
2 and
Feifei He
1,*
1
School of Agriculture, Yunnan University, Kunming 650500, China
2
Dehong Tropical Agriculture Research Institute, Dehong 678600, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(23), 13031; https://doi.org/10.3390/ijms252313031
Submission received: 29 October 2024 / Revised: 30 November 2024 / Accepted: 2 December 2024 / Published: 4 December 2024
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

WUSCHEL-related homeobox (WOX) genes play significant roles in plant development and stress responses. Difficulties in somatic embryogenesis are a significant constraint on the uniform seedling production and genetic modification of Coffea arabica, hindering efforts to improve coffee production in Yunnan, China. This study comprehensively analyzed WOX genes in three Coffea species. A total of 23 CaWOXs, 12 CcWOXs, and 10 CeWOXs were identified. Transcriptomic profile analysis indicated that about half of the CaWOX genes were actively expressed during somatic embryogenesis. The most represented CaWOXs were CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b, which are suggested to promote the induction and development of the embryogenic callus, whereas CaWOX13a and CaWOX13b are suggested to negatively impact these processes. Co-expression analysis revealed that somatic embryogenesis-related CaWOXs were co-expressed with genes involved in embryo development, post-embryonic development, DNA repair, DNA metabolism, phenylpropanoid metabolism, secondary metabolite biosynthesis, and several epigenetic pathways. In addition, qRT-PCR showed that four WOX genes responded to cold stress. Overall, this study offers valuable insights into the functions of CaWOX genes during somatic embryogenesis and under cold stress. The results suggest that certain WOX genes play distinct regulatory roles during somatic embryogenesis, meriting further functional investigation. Moreover, the cold-responsive genes identified here are promising candidates for further molecular analysis to assess their potential to enhance cold tolerance.

1. Introduction

WUSCHEL-related homeobox (WOX) proteins are named in reference to the Arabidopsis stem cell regulator, WUSCHEL, and belong to a plant-specific subgroup of the homeodomain (HD) superfamily [1,2,3]. The homeodomain of HD superfamily proteins comprises 60 amino acid residues that arrange themselves into a distinct “helix–loop–helix–turn–helix” three-dimensional configuration [4]. The second and third helical segments of the domain unite to form a “helix–turn–helix” motif, facilitating precise recognition and binding to specific DNA sequences [4,5]. Compared to other members of the HD superfamily, WOX proteins exhibit unique additional motifs, including FYWFQNH, FYWFQNR, and YNWFQNR, which correlate specifically with their phylogenetic groups (the WUS/modern, intermediate, and ancient clades, respectively) [2,6]. The ancient clade encompasses conserved WOX genes across diverse taxa, from algae to angiosperms, while the intermediate clade comprises members in plant groups like ferns to angiosperms. Lastly, members of the WUS/modern clade are exclusively present in seed plants [7].
Previous studies have demonstrated that WOX genes play diverse functional roles during plant developmental processes, including embryonic patterning, stem cell maintenance, flower architecture, and somatic embryogenesis [8,9]. For example, AtWUS defines stem cell and floral meristem identities, while also regulating maintenance of the shoot apical meristem (SAM) [1,10]. Overexpression of WUS genes in many plants, such as Arabidopsis, Gossypium hirsutum, and Coffea canephora, can increase their ability to form somatic embryos [11,12,13,14]. Numerous AtWOX genes and their corresponding functions have been characterized in Arabidopsis, providing an important framework for understanding their roles in plant development. AtWOX1 plays a pivotal role in meristem development through the regulation of CLV3 expression and SAMDC activity [15]. During embryo development, AtWOX8 and AtWOX9 redundantly affect basal and apical lineage development by influencing the expression of AtWOX2 and localizing the auxin response [16]. Notably, AtWOX2 serves as a marker for the apical cell lineage and is highly expressed in the egg cell and zygote, independent of exogenous auxin influence [16]. AtWOX3 is expressed in leaf and floral primordia margins, recruiting founder cells from meristem layers to form lateral domains in vegetative and floral organs [17]. Research has shown that AtWOX4 and AtWOX14 act redundantly to promote the proliferation and differentiation of procambial cells via distinct pathways. In particular, AtWOX4 interacts with PXY (Phloem intercalated with xylem) to augment auxin responsiveness in the cambium [18]. Conversely, AtWOX14 promotes vascular cell differentiation and lignification in the inflorescence stems of Arabidopsis by increasing the accumulation of bioactive gibberellin (GA) [19,20]. Under the control of Auxin Response Factor 10 (ARF10) and ARF16, AtWOX5 is expressed in the quiescent center (QC), functioning as a local, QC-specific regulator that activates TAA1-mediated auxin biosynthesis to regulate the functioning of the distal meristem [21,22]. During ovule development, AtWOX6 is expressed in the embryo, suspensor, and endosperm nuclei but is absent in the integument [23]. AtWOX7, which is activated by sugar and repressed by auxin, regulates lateral root development through the direct inhibition of the cell cycle gene CYCD6;1 [24]. CYCD6;1 can also be independently induced by auxin during asymmetric stem cell division in the root [25]. AtWOX11 serves as a direct target of the auxin signaling pathway, regulating founder cell establishment during de novo root regeneration and callus formation [26]. AtWOX12 acts redundantly with AtWOX11 and is involved in the first step of the cell fate transition during de novo root organogenesis in Arabidopsis [27]. Lastly, AtWOX13 is induced by auxin and regulates the cell fate determination of pluripotent callus to inhibit new shoot generation [28]. Thus, WOX genes contribute diverse roles to several developmental processes in Arabidopsis through their interactions with auxin and other homomers.
In addition to their roles in plant and somatic embryogenesis development, WOX genes are also involved in responses to abiotic stressors in many plant species, including cold, drought, and salt stress [29,30]. In Arabidopsis, AtWOX6 plays a role in freezing tolerance by affecting the activities of gene products that are independent of the C-repeat binding factor (CBF) pathway [31]. In addition to Arabidopsis, many WOX genes have been identified that respond to cold stress and other abiotic stressors in rice, pineapple (Ananas comosus L.), paper mulberry, soybean, and tea tree (Camellia sinensis) plants [32,33,34,35,36].
Coffea arabica L., known as Arabica coffee, belongs to the Rubiaceae family and accounts for over 70% of coffee bean production [37]. In China, over 98% of the cultivation and yield of C. arabica originates from the Yunnan province [38]. With the increase in demand for Arabica coffee beans, C. arabica cultivation has spread towards the highlands in Yunnan, where the temperature occasionally reaches 0 °C [39,40]. Thus, cold tolerance is a desired characteristic of C. arabica plants in this region, in addition to uniform clonal seedlings, since the yield and quality of traditional seedlings produced by seeds are unstable due to the allotetraploid (2n = 4x = 44 chromosomes) and autogamous nature of C. arabica. Therefore, understanding cold-responsive and somatic embryogenesis developmental mechanisms in C. arabica can assist in the development of cold-tolerant varieties, improve the efficiency of somatic embryogenesis, and shorten the somatic embryogenesis period.
The aim of the current study was to systematically identify WOX genes in C. arabica based on newly published genome data [41] and to reveal the cold-responsive and somatic embryogenesis developmental roles of Coffea arabica WUSCHEL-related homeobox (CaWOX) genes. Our results enhance our understanding of the roles of the CaWOX gene family in cold responses and somatic embryogenesis, thus potentially accelerating the molecular breeding programs for Coffea spp. plants in China.

2. Results

2.1. Identification and Phylogenetic Analysis of WOX Genes in Three Coffee Plants

Based on HMM and BLASTp searches, 23 CaWOXs, 12 CcWOXs, and 10 CeWOXs were identified in the genomes of C. arabica, C. canephora, and C. eugenioides, respectively (Table 1). The identified CaWOXs/CcWOXs/CeWOXs were named based on their best matching homologs in Arabidopsis. The number of WOXs identified in tetraploid C. arabica was comparable to the combined total found in its diploid progenitors, C. canephora and C. eugenioides. Similar to previous results from Arabidopsis, phylogenetic analysis suggested that the WOX proteins from Arabidopsis and coffee plants belonged to three clades including the WUS/modern, ancient, and intermediate-aged clades (Figure 1). The WUS clade was the largest group in this phylogenetic tree, containing 15, 8, 7, and 8 members from C. arabica, C. canephora, C. eugenioides, and Arabidopsis, respectively. Compared with AtWOXs, some WOX gene families, such as WOX1 and WOX13, were expanded in the three coffee plants, while WOX3 family expansion was only observed in C. arabica and C. canephora (Figure 1). WOX6, WOX7, WOX9, WOX10, WOX12, and WOX14 were not encoded by the three coffee plant genomes (Figure 1), potentially due to the whole-genome polyploidization event that occurred in the lineage leading to Arabidopsis, which diverges from the common ancestor shared with Coffea [42].

2.2. Gene Structure and Domain Analysis of CaWOXs

To explore WOX diversity, their gene structures and conserved motifs were analyzed. Most WOX genes, except WOX13 and WOX7, contained at least one intron, and two introns represented the most common gene structure configuration (Figure 2B). Members from the same WOX gene family exhibited similar gene structures, indicating that they potentially encoded conserved functions. Analysis of the conserved motifs indicated that 10 were identified in WOXs (Figure 2C). The 10 motifs of the typical WOX proteins followed the order of Motif 6–Motif 2–Motif 1–Motif 3–Motif 9–Motif 8–Motif 5–Motif 7–Motif 10–Motif 4. Motif 1 contained a homeodomain conserved sequence, but the other motifs did not share significantly conserved motifs based on BLAST searches in the National Center for Biotechnology Information (NCBI) and protein families (Pfam) databases (Figure 2C and Table S1). Furthermore, Motif 1 was found in all WOXs, while Motif 4 was only found in the ancient and intermediate clades, with no annotation target from the public protein database. Motif 9 was only found in Ca/Cc/CeWOX3 (Figure 2C and Table S1).

2.3. Expression Profiles of CaWOXs in Different Tissues

To evaluate the functions of CaWOXs during development, their expression patterns were compared using RNA-sequencing (RNA-seq) data from 14 organs or tissues, including roots, stems, leaves, meristems, flower buds, flowers, fruit perisperm (five samples), and 10-day imbibed seeds (three samples) (Table S2). CaWOXs were differentially expressed among the 14 tissue samples (Figure 3). CaWOX4a and CaWOX4b were predominantly expressed in roots and stems. CaWOX1c and CaWOX1d were mostly expressed in all tissues of 10-day imbibed seeds. CaWOX5a, CaWOX5b, CaWOX13c, and CaWOX13d were predominantly expressed in embryos of 10-day imbibed seeds. CaWOX8a, CaWOX8b, CaWOX11a, and CaWOX11b were mainly expressed in the embryo and micropylar endosperm of 10-day imbibed seeds. Furthermore, CaWOX13a and CaWOX13b were predominantly expressed in all tissues except the lateral endosperm of 10-day imbibed seeds (Figure 3).

2.4. Expression Patterns of CaWOX Genes During Somatic Embryogenesis

Previous research has reported that WOX genes play key roles during somatic embryogenesis [14]. To explore the functions of CaWOXs during somatic embryogenesis, the CaWOX expression patterns from leaf explants (L1) to globular embryos (E1) during 13 key developmental stages were re-calculated and re-analyzed from previous studies (Figure 4 and Table S3) [43]. The expression levels of CaWOX4a and CaWOX4b were mainly expressed from 1 to 5 weeks of explant dedifferentiation (D1–D3). CaWOX13a and CaWOX13b were highly expressed from leaf explants (L2) to primary callus (C1), while CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b were predominantly expressed from embryogenic callus (C2) to globular embryos (E1). Further, the expression levels of these eight CaWOXs were analyzed again with four samples (L2, D1, C2, and E1) during the 13 key developmental stages using qRT-PCR (Figure 5). Based on qRT-PCR results, CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b were highly expressed in C2 and E1 samples, while CaWOX4a, CaWOX4b, CaWOX13a, and CaWOX13b were highly expressed in D1 samples (Figure 5B and Table S4).
These results suggest that CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b promote the induction and development of the embryogenic callus, whereas CaWOX13a and CaWOX13b may have negative impacts on this process. To further identify the functions of CaWOX2a, CaWOX2b, CaWOX8a, CaWOX8b, CaWOX13a, and CaWOX13b during embryogenic callus induction and development, co-expression analysis was carried out using the RNA-seq datasets. Considering a PCC value of PCC < −0.9 or >0.9 as reflecting a strong correlation, CaWOX2a, CaWOX2b, CaWOX8a, CaWOX8b, CaWOX13a, and CaWOX13b were co-expressed with 1052, 407, 520, 500, 558, and 536 genes, respectively (Table S5). Subsequently, GO enrichment analysis was carried out to identify the biological processes related to these co-expressed genes (Figure 6). Embryo development, plant organ development, post-embryonic development, and post-embryonic organ development were represented by genes co-expressed with CaWOX2a and CaWOX2b (Figure 6). Root meristem growth, multicellular organismal process, and multi-organism reproductive process were among the categories of genes co-expressed with CaWOX8a and CaWOX8b (Figure 6). In addition, phenylpropanoid metabolic, secondary metabolite biosynthetic, and oxidation–reduction processes were represented by genes co-expressed with CaWOX13a and CaWOX13b (Figure 6). Furthermore, regulation of chromatin organization, regulation of chromosome organization, DNA repair, and DNA metabolic process were represented by genes co-expressed with CaWOX2a, CaWOX2b, and CaWOX8a (Figure 6). Histone modification, histone H3-K4 methylation, histone lysine methylation, chromatin assembly or disassembly, and the regulation of histone modification were functions related to the genes co-expressed with CaWOX2a (Figure 6).

2.5. Expression Patterns of CaWOX Genes Under Cold Stress and Other Stress Treatments

Cold stress can be a major natural disaster for Chinese coffee production. WOX genes have been reported to be responsive to cold stress [32,33]. The expression patterns of CaWOXs under cold stress were consequently analyzed with qRT-PCR using the two top pairs of mature leaves of one-year old C. arabica seedlings subjected to cold treatment. After cold acclimatization (CA), the tips of C. arabica leaves exhibited symptoms of frost damage, and under cold treatment (CT), these symptoms spread to cover half of the leaves (Figure 7A). In agreement with the symptoms observed on the leaves, electrical conductivity increased minimally under CA conditions, but increased by up to 67% under CT conditions (Figure 7B). The expressions of four CaWOXs were identified as responsive to cold treatment when considering thresholds of a fold-change exceeding two (Figure 7C–F). Specifically, CaWOX1c and CaWOX1d expression increased under both CA and CT conditions, whereas CaWOX13a expression decreased under both CA and CT conditions (Figure 7C–E). CaWOX13c initially demonstrated increased expression under CA conditions but subsequently returned to normal expression levels under CT conditions (Figure 7E,F).
In addition to cold stress, drought, elevated atmospheric CO2, and high-temperature stress are also considered among the most impactful stressors in global coffee production. To further evaluate the functions of CaWOXs of C. arabica in stress response, RNA-seq data for CaWOXs under drought stress, elevated temperatures stress, elevated air CO2 stress, and nitrogen stress were re-evaluated from previous studies [44,45,46]. Briefly, CaWOX1d exhibited higher expression in the drought-tolerant C. arabica cultivar IAPAR59 (I59) under both control and drought treatments (Figure 8). CaWOX3b was only expressed in the drought-tolerant cultivar (I59) after drought treatment, while CaWOX4b exhibited a response to drought only in the drought-susceptible cultivar (Rubi) (Figure 8). When exposed to elevated air CO2, CaWOX expression was not significantly different. However, CaWOX1c, CaWOX1d, CaWOX13c, and CaWOX13d exhibited weak expression responses to elevated temperatures under both ambient and elevated air CO2 concentrations (Figure 8). Under nitrogen stress, both CaWOX4a and CaWOX4b exhibited increased expression, but CaWOX4b displayed a lagged response (Figure 8). In addition, CaWOX4a and CaWOX4b were more highly expressed in the heat-sensitive cultivar Catuaí under both control and water conditions, in addition to being continuously expressed in the heat-tolerant cultivar Acauã under these conditions (Figure 8).

3. Discussion

3.1. WOX Genes in C. arabica

WOX genes are important in plant development, and thus, genome-wide analyses of the WOX gene family have been conducted in Arabidopsis, rice, cucumber, soybean, Rosa hybrida, Nelumbo nucifera, Medicago sativa, and tea (Camellia sinensis) plants [6,9,35,36,47,48,49]. In the present study, 23 WOX genes were identified in C. arabica, in addition to 12 members in C. canephora and 10 in C. eugenioides (Table 1). Previously, only seven members had been identified in C. arabica based on an in silico analysis of the expressed sequence tag database [50]. The number of WOXs isolated in the current study was significantly higher, owing to analysis of a newly published chromosome-level genome for C. arabica [41], leading to greater accuracy in CaWOX identification. Arabidopsis underwent genome polyploidization and gene loss events, resulting in a greater number of AtWOX genes compared to the number of CcWOXs and CeWOXs identified in Coffea plants (Table 1) [42,51].

3.2. CaWOX Functions

The evaluation of gene expression patterns can help to predict gene functions. Consequently, CaWOX gene expression profiles were analyzed from previously published datasets from 14 diverse tissues, in addition to expressional profiles responsive to drought, elevated atmospheric CO2, and high-temperature stress (Figure 3 and Figure 8). Some CaWOX genes exhibited tissue-specific expression, while others exhibited differential expression in response to drought, elevated atmospheric CO2 levels, and high temperatures. For example, homologs of AtWOX4, CaWOX4a, and CaWOX4b were predominantly expressed in roots and stems (Figure 3). In Arabidopsis, AtWOX4 confers auxin responsiveness to cambium cells and is responsible for extended root and stem thickening [18]. These results indicate that the function of WOX4 may be conserved among species and that CaWOX4 genes may confer functional redundancy. Furthermore, AtWOX11 has been reported to play a role during the induction of seed dormancy and release stages [52]. The high expression levels of CaWOX11a and CaWOX11b in the seed embryo and seed micropylar endosperm of imbibed seeds suggests similar functioning (Figure 3). These results collectively offer new insights for future functional prediction and characterization of CaWOXs.

3.3. CaWOX and Somatic Embryogenesis

Somatic embryogenesis (SE) is a micropropagation technique through which plants can regenerate bipolar structures from somatic cells, thus producing genetically uniform seedlings. In China, achieving embryonic competence in callus tissue remains a formidable challenge in the process of somatic embryogenesis for C. arabica, thereby impeding the progress of the coffee industry. Understanding the genes that respond during the SE process can offer valuable insights for accelerating it [53]. Previous studies have demonstrated that the overexpression of WOX genes, such as AtWUS, TaWOX5, and ZmWUS2, can induce somatic embryogenesis-related genes and promote spontaneous regeneration [54,55,56]. In C. arabica, a WOX-like gene belonging to the intermediate clade has been shown to be related to the embryogenic process [50], while other members remain unstudied. In this study, comprehensive investigation and characterization of CaWOXs in somatic embryogenesis was conducted via genome-wide analysis. Further, the expression levels of highly related genes were confirmed using samples from key stages, ranging from explants to torpedo-shaped embryos (Figure 4 and Figure 5). Unlike previous studies in Arabidopsis and maize [55,57], CaWUS was not expressed during C. arabica somatic embryogenesis, while CaWOX2s and CaWOX8s were markedly increased in expressed during the transformation from primary (non-embryonic callus) to embryonic callus, indicating that these genes play vital roles in the key embryonic transition process (Figure 4). In cotton, the WOX8 and WOX2 genes play similar roles instead of WUS, or they merely function in a redundant manner [14].
Considering the roles of WOX2 and WOX8 during somatic embryogenesis, a previous study has shown that PpWOX2 (Pinus pinaster WOX2, a homolog of AtWOX2) promotes somatic embryogenesis and organogenesis in Arabidopsis [58]. In Arabidopsis, WOX8 and WOX9 are expressed at very early stages of embryo development, and ectopic co-expression of WOX2 with either WOX8 or WOX9 enhances regeneration from leaf segments and free cells in Nicotiana tabacum, suggesting that they may be critical regulators of somatic embryogenesis [59,60]. In woody plants, the expression patterns of WOX2, WOX8, and WOX9 indicate their involvement in regulating somatic embryogenesis [61]. In C. arabica, no orthologs of AtWOX9 were identified. However, CaWOX2s and CaWOX8s exhibited high expression levels from the embryogenic callus stage to the torpedo-shaped embryo stage, indicating their roles as crucial regulators of somatic embryonic properties (Figure 4).
In addition to CaWOX2s and CaWOX8s, CaWOX13a and CaWOX13b were responsive to the transformation process from non-embryonic to embryonic callus, but they exhibited decreased expression during this stage (Figure 4). In Arabidopsis, AtWOX13 suppresses de novo shoot regeneration from callus and impacts regeneration efficiency in Arabidopsis [28], suggesting that AtWOX13 and its homologs in other plants play negative roles during the key embryonic transition process.

3.4. Potential Mechanisms of CaWOXs in Somatic Embryogenesis

Although WOXs are associated with somatic embryogenesis, their specific functions remain largely unknown. Co-expression analysis can offer potential insights into the roles of their target genes. For example, co-expression and GO enrichment analysis have previously highlighted numerous biological processes, encompassing post-embryonic development, reproductive processes in multicellular organisms, and other processes associated with somatic embryogenesis in plants [62,63]. In C. arabica, post-embryonic development, root meristem growth, and multicellular organismal processes were implicated as functions of co-expressed genes of the CaWOX2 and CaWOX8 genes (Figure 6), implying their roles in somatic embryogenesis for promoting calluses towards embryogenesis and contributing to the development of roots, shoots, and leaf organs.
Epigenetic modifications play pivotal roles in the signaling cascade that leads to alterations in cell genetic programming, thereby initiating somatic embryo development [64,65]. The regulation of epigenetic mechanisms has recently emerged as a highly promising strategy for improving somatic embryogenesis in plants. Such regulation can be orchestrated through processes such as DNA methylation, chromatin remodeling, and small-RNA-mediated regulation [57,66]. For example, LEC2 (Leafy Cotyledon 2) can be rapidly induced by auxin through chromatin remodeling. Subsequently, activated LEC2 promotes the expression of WOX2 and WOX3, thereby facilitating somatic embryo formation [64]. In C. arabica, the regulation of chromatin organization, histone modification, histone H3-K4 methylation, histone lysine methylation, and other epigenetic processes was implicated as functions of genes co-expressed with the CaWOX2 and CaWOX8 genes (Figure 6), suggesting that CaWOX genes may also regulate somatic embryogenesis by epigenetic means.
CaWOX13a and CaWOX13b function as negative regulators for embryonic callus formation, and secondary metabolite biosynthetic processes were implicated as functions among the genes that were co-expressed with them (Figure 4, Figure 5 and Figure 6). In cotton, secondary metabolite contents in the embryonic callus were less abundant than in a non-embryonic callus [67]. Phenylpropanoid metabolism may reduce oxidative stress in non-embryonic callus and promote embryogenic capacity [43,68,69]. Here, the phenylpropanoid biosynthesis pathway was represented by CaWOX13a and CaWOX13b that were highly expressed in explants from the dedifferentiation stage to the primary callus stage (Figure 4, Figure 5 and Figure 6). Taken together, these results demonstrate that CaWOX participates in or regulates somatic embryogenesis through a multitude of biological processes.

3.5. CaWOX and Cold Stress

Low temperatures can cause irreversible damage to plants and adversely affect their growth and development. However, many plants enhance their cold tolerance through a process known as cold acclimation (CA) [70]. During cold stress, the expression of cold-regulated (COR) genes is induced, as controlled by various transcription factors (TFs). This induction leads to changes in the physiological and biochemical characteristics of the plant. Among these TFs, C-repeat binding factors (CBFs) are particularly well represented [71]. In Arabidopsis, AtWOX6 (also known as HOS9) can enhance cold tolerance by affecting gene activity independent of the CBF pathway [31]. WOX6 was found to be inducible by auxin, while cold stress can affect auxin transport and intracellular auxin gradients, suggesting potential links among WOXs, auxin, and cold-stress responses in the regulation of plant growth and development [72,73]. However, these links are still poorly understood [74]. In C. arabica, no orthologs of AtWOX6 were identified (Figure 1). However, its closest homologs, CaWOX1c and CaWOX1d, were more highly expressed under both CA and CT conditions (Figure 7), indicating that CaWOX1c/d may act in similar roles as WOX6. In pineapple plants, most WOX genes were decreased in expression by cold treatment, indicating that some WOXs may exert negative roles during cold tolerance [33]. For C. arabica, one gene (CaWOX13a) was more highly expressed in response to both CA and CT, while another gene (CaWOX13c) was only more highly expressed in response to CA (Figure 7), indicating that these genes play vital roles in cold-stress responses.

4. Materials and Methods

4.1. Identification of WOX Genes in Coffea Species

The entire genome sequences of C. arabica, C. canephora, and C. eugenioides were retrieved from the Online Resource for Community Annotation of Eukaryotes (ORCAE, https://bioinformatics.psb.ugent.be/orcae/overview/Coara, Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium, accessed on 16 August 2023), Coffee Genome Hub (http://coffee-genome.org, accessed on 16 August 2023), and NCBI (https://www.ncbi.nlm.nih.gov/genome, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD USA, accessed on 16 August 2023), respectively [41,42]. The protein sequences of 15 AtWOXs were retrieved from The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org, Phoenix Bioinformatics Corporation, Newark, CA, USA, accessed on 16 August 2023) [9]. These 15 AtWOX protein sequences were used as queries to perform a BLASTp search (E-value < 1 × 10−5) against all of the protein sequences of the three coffee plants. Candidate protein sequences of the three Coffea plants identified from BLASTp searches were used as queries to search against the Pfam HMM library (Pfam 34.0) [75,76] using a Hidden Markov Model (HMM) profile (version 3.1.b2). Search hits with a Homeobox domain (PF00046) were identified and considered WOX family candidates based on an E-value cutoff of 0.001. The amino acid sequences of the WOX candidates were analyzed to determine the presence of three unique motifs (FYWFQNH, FYWFQNR, and YNWFQNR) previously defined for WOXs [6]. Candidates without unique motifs were not considered further. All Coffea arabica WUSCHEL-related homeobox (CaWOX) genes were identified based on their best hit among Arabidopsis WOX proteins.

4.2. Phylogenetic and Bioinformatic Analysis of CaWOXs

Multiple alignments of the WOX protein sequences from the three coffee plants and Arabidopsis were performed using the MUSCLE program implemented in MEGA6 with default parameters [77]. Unrooted phylogenetic trees were constructed using MEGA6 with neighbor-joining methods and analysis parameters including 1000 bootstrap replicates, and the Jones–Taylor–Thornton (JTT) substitution model [78]. The isoelectric points (PIs) and molecular weights (MWs) of the CaWOXs were analyzed using the ProtParam tool (Expasy, the Swiss Bioinformatics Resource Portal, https://web.expasy.org/protparam, accessed on 16 August 2023) [79]. The exon–intron gene structures of CaWOXs were represented using the Gene Structure Display Server (GSDS, version 2.0, http://gsds.gao-lab.org, accessed on 16 August 2023) based on the genomic General Feature Format (GFF) annotation file [80]. Conserved motifs were identified using the MEME software program (version 5.5.2, http://meme-suite.org, accessed on 16 August 2023) [81] based on the default settings, and the results were analyzed using TBtools [82]. The identified motifs were annotated using comparisons against the InterPro (https://www.ebi.ac.uk/interpro, accessed on 16 August 2023) [83] and NCBI databases.

4.3. Cold Treatment, Electrolyte Leakage Test, and Tissue Culture

The 1-year-old seedlings of C. arabica (Catimor, CIFC7963) were subjected to cold treatment. Cold treatments were carried out as described previously [84]. For the control group (CK), the plants were subjected to a temperature of 24 °C during the day and 20 °C at night for 7 days in a growth chamber. This was followed by another 7 days at 13 °C during the day and 8 °C at night to induce cold acclimation (CA). Subsequently, the plants were exposed to 4 °C both during the day and at night for 3 days (CT). Throughout the cold treatment, the environmental conditions were maintained at a humidity of 60%, a luminosity of 600–650 μmol m−2s−1, and a photoperiod of 16 h light and 8 h dark. At the end of each treatment period, five plants were selected, and the two most recently matured pairs of leaves were collected from the top of the plants. These leaves were immediately frozen in liquid nitrogen and stored at −80 °C for later analysis. The entire cold treatment process was repeated three times independently with different sets of plants.
Immediately after cold treatment, electrolyte leakage was measured in cold-treated and control plants as previously described [85], with some modifications. Briefly, five leaf discs were randomly picked from a mix of discs (10 discs per plant from the 5 most recently fully expanded leaves that were mixed together). These discs were placed in a glass tube containing 10 mL of distilled water. The samples were then incubated on an orbital shaker at 150 rpm for 30 min at room temperature. Then, initial conductivity (I) was measured using a CON110 conductivity meter (Oakton Ins., Vernon Hills, USA). Next, the leaf discs were boiled in water for 10 min and cooled to room temperature, and the final conductivity (F) was measured. The relative electrolyte leakage was calculated using the following formula: I/F×100.
For tissue culture, the second pair of fully expanded leaves (from top to bottom) was collected from two-year-old coffee seedlings (C. arabica L. cv. ‘Catimor CIFC 7963’). These leaves were used as explants. The coffee seedlings were grown under greenhouse conditions at Yunnan University in the spring of 2021, as follows: 16 h light/8 h dark, 60% humidity, 600–650 µmol m−2 s−1 light intensity, and 24/20 °C (day/night). The explant leaves were sterilized with 70% alcohol and 1% sodium hypochlorite. After sterilization, the explants were washed 4 or 5 times with distilled water and dissected into 0.5 cm2 blocks. The blocks were then placed on half-strength Murashige and Skoog (MS) solid medium supplemented with 1 mg/L 6-Benzylaminopurine (6-BA) and 2 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) for callus induction. After 2 months of cultivation, the induced callus was transferred to embryo induction medium, which contained 0.5 mg/L 1-Naphthaleneacetic acid (NAA), 5 mg/L 6-BA, and 4 mg/L AgNO3. After 6, 7, and 9 months of cultivation, the compact primary callus, embryogenic callus, and globular embryos were collected for further analysis and frozen immediately in liquid nitrogen, followed by storage at −80 °C until subsequent use.

4.4. Expression Analysis of CaWOX Genes Within RNA-Seq Data

To analyze CaWOX expression among tissues, publicly available RNA-seq data from 14 C. arabica organs or tissues were retrieved from the NCBI database via the BioProject accessions PRJNA339585, PRJNA305756, and PRJNA5546 [10,86]. Data from tissues comprising roots, stems, leaves, meristems, flower buds, flowers, fruit perisperm (5 samples), and 10-day imbibed seeds (3 samples) were included. The sequencing reads were mapped to the new published reference genome (“ET-39”) using the HISAT2 (version 2.1.0) software program [41,87]. Gene expression levels were re-calculated using the transcripts per million (TPM) metric, and heatmaps for CaWOX expression profiles were generated using the TBtools software program (version 1.0987663) [83].
The expression of CaWOX involved in the somatic embryogenesis (SE) developmental process was recalculated from a previous study (BioProject PRJNA744419) using the TPM metric [43]. Thirteen sampling stages were selected to encompass the entire somatic embryogenesis (SE) process, ranging from leaf explant initiation to the torpedo-shaped embryo development, as demonstrated in previous studies [43,88]. Leaves collected from greenhouse plants (designated L1), explants undergoing dedifferentiation at 1 (D1), 2 (D2), and 5 weeks (D3), compact primary callus obtained 3 months post-induction (C1), embryogenic callus harvested 7 months post-induction (C2), established cell clusters after 4 months in liquid proliferation medium (C3), pro-embryogenic masses at various stages in redifferentiation medium 1 week after auxin withdrawal (R1), 24 h (R2), 72 h (R3), and 10 days after reducing cell density (R4)], and globular embryos developed after 3 weeks of culture (E1) were sampled.
The TPM values of CaWOX genes were also recalculated after drought, elevated air CO2, nitrogen stress, and elevated temperature treatments, as described in previous studies (BioProjects PRJNA282394, PRJNA606444, PRJEB15539, and PRJNA609253) [44,45,89,90]. The drought stress experiment was conducted using two contrasting Coffea arabica cultivars: Rubi MG1192 (Rubi, drought-susceptible) and IAPAR59 (I59, drought-tolerant). Samples of the shoot apices were collected for RNA-seq analysis [44]. The treatment involving elevated atmospheric CO2 was conducted using the 1.5-year-old Icatu variety, with CO2 concentrations set at 380 μmol mol−1 (ambient CO2 or aCO2) and 700 μmol mol−1 (elevated CO2 or eCO2). The newly matured leaves from plagiotropic and orthotropic branches were collected for RNA-seq [90]. In nitrogen-stress experiments, the lateral roots of the IAPAR59 variety were exposed to nitrogen-free conditions for 0, 1, and 10 days [89]. Elevated temperature treatment was carried out using two Coffea arabica genotypes, cvs. Acauã and Catuaí IAC 144, which have been suggested to differ in heat tolerance [45].

4.5. Co-Expression and GO Enrichment Analysis

Candidate CaWOXs were used as target genes for genome-wide co-expression analysis to identify genes based on the expression profiling of C. arabica RNA-seq data during somatic embryogenesis [43]. A cutoff threshold of <−0.9 or >0.9 was used for Pearson correlation coefficient (PCC) analysis. Gene Ontology (GO) enrichment analysis was performed using agriGO with a false discovery rate (FDR) value below 0.05 [91], and the top 20 GO enrichment items were visualized using R packages.

4.6. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR

Following the manufacturer’s instructions, total RNA was isolated from homogenized samples using the RNAiso Plus Reagent (Takara Biomedical Technology Co., Ltd., Beijing, China). First-strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara Biomedical Technology Co., Ltd., Beijing, China) using 1 μg of total RNA. The synthesized cDNA was diluted to 10 ng/μL for PCR. Quantitative real-time PCR (qRT-PCR) was conducted using the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems®, Foster City, CA, USA), with the following reaction system specifications: a total volume of 20 µL, consisting of 2 µL of cDNA, 1.0 µL of each primer at a 10 µM concentration, 10 µL of SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (TAKARA BIO INC., Shiga, Japan), and 6 µL of distilled water. The thermal cycling protocol for PCR involved an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and extension at 56 °C for 30 s. The PCR results were analyzed using the 2−∆∆CT method, and all primer sequences used are listed in Table S6.

5. Conclusions

In this study, genome-wide identification of WOX genes in three Coffea species was conducted. A total of 45 WOX genes were identified classified into three clades, consistent with previous studies. During somatic embryogenesis in C. arabica, only half of the WOX genes were actively expressed, with the expression patterns indicating that different WOX gene family members might function in different somatic embryogenesis stages of C. arabica. CaWOX2s, CaWOX8s, CaWOX13a, and CaWOX13b emerged as potential key genes that could play important roles in somatic embryogenesis. Co-expression analysis further suggested various specific biological processes that CaWOXs may be involved in during somatic embryogenesis. Additionally, four CaWOX genes were identified that responded to cold stress via altered expression levels. The comprehensive analysis of the CaWOX gene family in this study and its roles in both C. arabica somatic embryogenesis and stress response offers valuable insights to promote future functional experimental studies.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms252313031/s1.

Author Contributions

Data curation, M.J., J.G. and Y.T.; Formal analysis, X.C. and X.Y.; Funding acquisition, F.H.; Investigation, L.W., D.J. and Z.X.; Methodology, X.B.; Resources, Z.X., X.B. and F.H.; Supervision, X.B. and F.H.; Writing—original draft, J.G. and X.D.; Writing—review and editing, X.D. and D.J. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the Applied Basic Research Project of Yunnan (202401AT070481), the National Natural Science Foundation of China (72261147759) and the Bill & Melinda Gates Foundation (BMGF, No. 2022YFAG1004), and the Scientific Research Fund Project of Yunnan Provincial Education Department (2023Y0230).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors would like to acknowledge the assistance of ERNIE Bot for initial grammar checking. All authors have read and approved the final version of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic relationships of WOX proteins from three coffee plants and Arabidopsis. Different clades are indicated by the background colors and branch lines.
Figure 1. Phylogenetic relationships of WOX proteins from three coffee plants and Arabidopsis. Different clades are indicated by the background colors and branch lines.
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Figure 2. Phylogenetic reconstruction (A), gene structures (B), and MEME motifs (C) for WOXs identified in three coffee plants and Arabidopsis. (B) Dark blue boxes represent exons and black lines indicate introns. (C) Colored boxes indicate motifs, as shown in the legend on the right.
Figure 2. Phylogenetic reconstruction (A), gene structures (B), and MEME motifs (C) for WOXs identified in three coffee plants and Arabidopsis. (B) Dark blue boxes represent exons and black lines indicate introns. (C) Colored boxes indicate motifs, as shown in the legend on the right.
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Figure 3. Expression of CaWOX genes in different tissues and organs of Coffea arabica based on RNA-sequencing data. Expression data were treated with log2(TPM+1) normalization, as indicated by the scale to the right. DAF, day after flowering. E, seed embryo. LE, seed lateral endosperm. ME, seed micropylar endosperm.
Figure 3. Expression of CaWOX genes in different tissues and organs of Coffea arabica based on RNA-sequencing data. Expression data were treated with log2(TPM+1) normalization, as indicated by the scale to the right. DAF, day after flowering. E, seed embryo. LE, seed lateral endosperm. ME, seed micropylar endosperm.
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Figure 4. Expression of CaWOX genes during somatic embryogenesis in Coffea arabica based on RNA-sequencing data. Expression data were treated with log2(TPM + 1) normalization, as indicated by the scale to the right. L1, leaves from plants. L2, D1, D2, and D3, leaf explants during dedifferentiation after 0 h, 1 week, 2 weeks, and 5 weeks, respectively. C1, primary callus. C2, embryogenic callus. C3, cell clusters obtained from proliferation medium. R1, cell clusters after induction in DIF (redifferentiation) medium for 1 week. R2, R3, and R4, cell clusters after 24 h, 72 h, and 10 d of reducing cell density, respectively. E1, globular embryos. E2, torpedo-shaped embryos.
Figure 4. Expression of CaWOX genes during somatic embryogenesis in Coffea arabica based on RNA-sequencing data. Expression data were treated with log2(TPM + 1) normalization, as indicated by the scale to the right. L1, leaves from plants. L2, D1, D2, and D3, leaf explants during dedifferentiation after 0 h, 1 week, 2 weeks, and 5 weeks, respectively. C1, primary callus. C2, embryogenic callus. C3, cell clusters obtained from proliferation medium. R1, cell clusters after induction in DIF (redifferentiation) medium for 1 week. R2, R3, and R4, cell clusters after 24 h, 72 h, and 10 d of reducing cell density, respectively. E1, globular embryos. E2, torpedo-shaped embryos.
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Figure 5. Expression patterns of CaWOX genes related to somatic embryogenesis. (A), four key stages selected for qRT-PCR analysis. L2 and D1, leaf explants during dedifferentiation after 0 h and 1 week, respectively. C2, embryogenic callus. E1, globular embryos. Bar = 2 mm. (B), qRT-PCR results of CaWOXs related to somatic embryogenesis within four key stages. Expression levels were represented with the method of 2−∆∆CT.
Figure 5. Expression patterns of CaWOX genes related to somatic embryogenesis. (A), four key stages selected for qRT-PCR analysis. L2 and D1, leaf explants during dedifferentiation after 0 h and 1 week, respectively. C2, embryogenic callus. E1, globular embryos. Bar = 2 mm. (B), qRT-PCR results of CaWOXs related to somatic embryogenesis within four key stages. Expression levels were represented with the method of 2−∆∆CT.
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Figure 6. Gene Ontology (GO) enrichment analysis of genes co-expressed with CaWOX2a, CaWOX2b, CaWOX8a, CaWOX8b, CaWOX13a, and CaWOX13b which involved in somatic embryogenesis. FDR, false discovery rate.
Figure 6. Gene Ontology (GO) enrichment analysis of genes co-expressed with CaWOX2a, CaWOX2b, CaWOX8a, CaWOX8b, CaWOX13a, and CaWOX13b which involved in somatic embryogenesis. FDR, false discovery rate.
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Figure 7. Phenotypic analysis of Coffea arabica seedlings after cold treatment and the relative expression patterns of CaWOX genes after cold treatment. (A), phenotype of Coffea arabica leaves after cold treatment. (B), electrolyte leakage of Coffea arabica leaves after cold treatment. (C–F), relative expression levels of cold-responsive CaWOXs based on qRT-PCR results. CK, control condition, the C. arabica seedlings were grown at 24/20 °C (day/night). CA, cold acclimatization, seedlings were grown at 13/8 °C (day/night). CT, cold treatment, seedlings were exposed to 4/4 °C (day/night).
Figure 7. Phenotypic analysis of Coffea arabica seedlings after cold treatment and the relative expression patterns of CaWOX genes after cold treatment. (A), phenotype of Coffea arabica leaves after cold treatment. (B), electrolyte leakage of Coffea arabica leaves after cold treatment. (C–F), relative expression levels of cold-responsive CaWOXs based on qRT-PCR results. CK, control condition, the C. arabica seedlings were grown at 24/20 °C (day/night). CA, cold acclimatization, seedlings were grown at 13/8 °C (day/night). CT, cold treatment, seedlings were exposed to 4/4 °C (day/night).
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Figure 8. Expression patterns of CaWOX genes under different stress treatments based on RNA-sequencing data. Rubi, drought-susceptible C. arabica cultivar Rubi MG1192. I59, drought-tolerant C. arabica cultivar IAPAR59. C, control. D, drought treatment. aCO2, ambient air CO2 (380 µL L−1). eCO2, elevated air CO2 (700 µL L−1). The terms 0 D, 1 D, and 10 D indicate 0, 1, and 10 days after nitrogen starvation. Acauã, C. arabica cultivar Acauã. Catuaí, C. arabica cultivar Catuaí IAC 144.
Figure 8. Expression patterns of CaWOX genes under different stress treatments based on RNA-sequencing data. Rubi, drought-susceptible C. arabica cultivar Rubi MG1192. I59, drought-tolerant C. arabica cultivar IAPAR59. C, control. D, drought treatment. aCO2, ambient air CO2 (380 µL L−1). eCO2, elevated air CO2 (700 µL L−1). The terms 0 D, 1 D, and 10 D indicate 0, 1, and 10 days after nitrogen starvation. Acauã, C. arabica cultivar Acauã. Catuaí, C. arabica cultivar Catuaí IAC 144.
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Table 1. Gene information for WOX from three coffee plants. bp, base pair; a.a., amino acid; MW, molecular weight; Kda, kilodalton; pI, isoelectric point.
Table 1. Gene information for WOX from three coffee plants. bp, base pair; a.a., amino acid; MW, molecular weight; Kda, kilodalton; pI, isoelectric point.
Gene NameGene IDChromosome LocationgDNA Length (bp)Protein Length (a.a.)MW (KDa)Isoelectric Point (pI)Best BLAST Hit Within Arabidopsis
At_IDAt_NameE-Value
CaWOX3aCara001g022170Chr 01119921624.889.35AT2G28610.1AtWOX35.50 × 10−46
CaWOX2aCara001g022960Chr 01136026129.45 8.79AT5G59340.1AtWOX29.46 × 10−52
CaWOX3bCara002g013730Chr 02119721624.87 9.35AT2G28610.1AtWOX33.45 × 10−43
CaWOX3cCara002g013760Chr 02119721624.87 9.35AT2G28610.1AtWOX33.45 × 10−43
CaWOX2bCara002g014580Chr 02136926229.44 9.05AT5G59340.1AtWOX22.33 × 10−49
CaWOX8bCara003g039800Chr 03209740745.00 8.75AT5G45980.1AtWOX81.39 × 10−78
CaWOX5aCara003g048620Chr 0365617419.70 8.76AT3G11260.1AtWOX52.89 × 10−60
CaWOX5bCara004g007900Chr 0466617419.70 8.76AT3G11260.1AtWOX52.89 × 10−60
CaWOX8aCara004g016390Chr 04209240744.89 8.55AT5G45980.1AtWOX85.16 × 10−79
CaWOX1aCara007g007070Chr 07216036240.90 6.18AT3G18010.1AtWOX15.26 × 10−60
CaWOX13dCara007g012080Chr 0774124728.26 5.43AT4G35550.1AtWOX132.86 × 10−49
CaWOX13cCara008g014990Chr 0874124728.26 5.54AT4G35550.1AtWOX132.31 × 10−49
CaWOX1bCara008g019840Chr 08216436240.97 6.53AT3G18010.1AtWOX13.10 × 10−59
CaWOX11aCara011g029950Chr 11241225727.90 5.61AT3G03660.1AtWOX112.40 × 10−63
CaWOX11bCara012g008700Chr 12241025727.90 5.61AT3G03660.1AtWOX112.40 × 10−63
CaWUSbCara013g011100Chr 13161128632.12 6.45AT2G17950.1AtWUS2.41 × 10−46
CaWOX13bCara013g012460Chr 13311928031.00 5.56AT4G35550.1AtWOX139.66 × 10−87
CaWOX13aCara014g017290Chr 14360427830.89 5.56AT4G35550.1AtWOX139.35 × 10−89
CaWUSaCara014g018710Chr 14161228632.14 6.45AT2G17950.1AtWUS2.03 × 10−46
CaWOX4aCara019g006470Chr 19112222225.44 9.51AT1G46480.1AtWOX41.00 × 10−76
CaWOX4bCara020g021370Chr 20107822225.47 9.51AT1G46480.1AtWOX41.36 × 10−76
CaWOX1dCara021g016240Chr 21152633738.23 7.15AT3G18010.1AtWOX11.03 × 10−31
CaWOX1cCara022g010550Chr 22152633738.26 6.84AT3G18010.1AtWOX19.18 × 10−32
CcWOX11Cc00g05100Chr un250325127.45 5.84AT3G03660.1AtWOX119.05 × 10−65
CcWOX3aCc00g26230Chr un119821524.93 9.51AT2G28610.1AtWOX32.71 × 10−45
CcWOX3bCc01g11960Chr 01119121524.87 9.57AT2G28610.1AtWOX38.83 × 10−42
CcWOX2Cc01g12690Chr 01135923726.88 8.93AT5G59340.1AtWOX23.43 × 10−52
CcWOX5Cc02g06840Chr 0266617319.70 8.76AT3G11260.1AtWOX52.79 × 10−60
CcWOX8Cc02g14220Chr 02208740645.00 8.75AT5G45980.1AtWOX81.35 × 10−78
CcWOX1aCc04g06330Chr 04325236140.97 6.05AT3G18010.1AtWOX13.52 × 10−60
CcWOX13bCc04g10680Chr 0474024628.26 5.43AT4G35550.1AtWOX132.77 × 10−49
CcWUSCc07g10660Chr 07160928532.09 6.45AT2G17950.1AtWUS2.66 × 10−46
CcWOX13aCc07g11890Chr 07381927931.00 5.56AT4G35550.1AtWOX139.32 × 10−87
CcWOX4Cc10g04700Chr 10238422125.47 9.51AT1G46480.1AtWOX41.31 × 10−76
CcWOX1bCc11g08460Chr 11152533638.23 7.15AT3G18010.1AtWOX11.01 × 10−31
CeWOX2LOC113774651Chr 1142926029.35 9.05AT5G59340AtWOX21.22 × 10−50
CeWOX3LOC113774781Chr 1119621524.87 9.35AT2G28610AtWOX33.34 × 10−43
CeWOX4LOC113749539Chr 10148222125.47 9.51AT1G46480AtWOX41.31 × 10−76
CeWOX1aLOC113753065Chr 11236233638.27 7.6AT3G18010AtWOX19.75 × 10−32
CeWOX5LOC113755443Chr 266317319.70 8.76AT3G11260AtWOX52.79 × 10−60
CeWOX8LOC113764000Chr 2240639243.19 8.25AT5G45980AtWOX81.04 × 10−78
CeWOX1bLOC113768485Chr 4300636140.97 6.53AT3G18010AtWOX13.01 × 10−59
CeWOX13aLOC113769015Chr 479726530.71 5.66AT4G35550AtWOX134.76 × 10−49
CeWOX13bLOC113778647Chr 7355727130.26 5.56AT4G35550AtWOX132.39 × 10−86
CeWOX11LOC113758086Chr un215525127.45 5.84AT3G03660AtWOX119.05 × 10−65
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Dong, X.; Gao, J.; Jiang, M.; Tao, Y.; Chen, X.; Yang, X.; Wang, L.; Jiang, D.; Xiao, Z.; Bai, X.; et al. The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response. Int. J. Mol. Sci. 2024, 25, 13031. https://doi.org/10.3390/ijms252313031

AMA Style

Dong X, Gao J, Jiang M, Tao Y, Chen X, Yang X, Wang L, Jiang D, Xiao Z, Bai X, et al. The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response. International Journal of Molecular Sciences. 2024; 25(23):13031. https://doi.org/10.3390/ijms252313031

Chicago/Turabian Style

Dong, Xiangshu, Jing Gao, Meng Jiang, Yuan Tao, Xingbo Chen, Xiaoshuang Yang, Linglin Wang, Dandan Jiang, Ziwei Xiao, Xuehui Bai, and et al. 2024. "The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response" International Journal of Molecular Sciences 25, no. 23: 13031. https://doi.org/10.3390/ijms252313031

APA Style

Dong, X., Gao, J., Jiang, M., Tao, Y., Chen, X., Yang, X., Wang, L., Jiang, D., Xiao, Z., Bai, X., & He, F. (2024). The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response. International Journal of Molecular Sciences, 25(23), 13031. https://doi.org/10.3390/ijms252313031

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