Identification and expression analysis of PUB genes in tea plant exposed to anthracnose pathogen and drought stresses

ABSTRACT

The plant U-box (PUB) gene family, one of the major ubiquitin ligase families in plants, plays important roles in multiple cellular processes including environmental stress responses and resistance. The function of U-box genes has been well characterized in Arabidopsis and other plants. However, little is known about the tea plant (Camellia sinensis) PUB genes. Here, 89 U-box proteins were identified from the chromosome-scale referenced genome of tea plant. According to the domain organization and phylogenetic analysis, the tea plant PUB family were classified into ten classes, named Class I to X, respectively. Using previously released stress-related RNA-seq data in tea plant, we identified 34 stress-inducible CsPUB genes. Specifically, eight CsPUB genes were expressed differentially under both anthracnose pathogen and drought stresses. Moreover, six of the eight CsPUBs were upregulated in response to these two stresses. Expression profiling performed by qRT-PCR was consistent with the RNA-seq analysis, and stress-related cis-acting elements were identified in the promoter regions of the six upregulated CsPUB genes. These results strongly implied the putative functions of U-box ligase genes in response to biotic and abiotic stresses in tea plant.

KEYWORDS:

• U-box
• ligase
• Camellia
• anthracnose
• drought

Introduction

Ubiquitination is one of the major types of post-translational modification (PTM) of proteins in eukaryotes. The ubiquitination pathway, involving the ubiquitin bond to a lysine residue in the target protein, requires three types of enzymes. The E1 activating enzyme is required to recruit and activate ubiquitin, and then the activated ubiquitin is delivered to the E2 conjugating enzyme, and finally the ubiquitin is transferred to the target protein for ubiquitination in presence of the E3 ubiquitin ligase.¹ Among these three types of enzymes, the E3 ligases are the largest family and serve as the central players for specifically recognizing the target substrates.^2,3 Based on domain organization and function, plant E3 ligases are mainly classified into three families, namely HECT (homologous to E6-associated protein C terminus) ligases,⁴ RING (really interesting new gene) ligases⁵ and U-box ligases.⁶

The U-box ligases are a class of family with a conserved U-box domain containing approximately 70 amino acids that were first identified in the yeast UFD2 (ubiquitin fusion degradation protein-2).⁷ The U-box shares great structural similarities with RING domain, while the major difference between these two domains is that the U-box lacks the classical zinc-binding ligand.⁸ The U-box-containing genes are widely distributed in eukaryotic species. However, compared to the fewer genes in yeast and animals, the plant U-box (PUB) genes are evolutionarily expanded to a larger family with more members. There are only 2 and 11 U-box genes in yeast^7,9 and mouse,¹⁰ respectively, while 63 PUBs identified in Arabidopsis thaliana,^11,12 67 in barley (Hordeum vulgare),¹³ 77 in rice,¹⁴ 91 in banana (Musa acuminata),¹⁵ 99 in Brassica oleracea¹⁶ and 125 in soybean.¹⁷ The large expansion of U-box proteins in plants implies that PUBs may function in a wide range of cellular processes unique to plants. It is supported by emerging evidences that PUBs act as positive or negative regulators in diverse biological processes including plant response and adaptation to biotic and abiotic stresses. Rice OsPUB44 positively regulates PAMP-triggered immunity, and its E3 ligase activity is restrained by its direct interactor XopP_Xoo, one of the effectors of rice pathogen Xanthomonas oryzae pv. oryzae.¹⁸ Similarly, potato StUBK, a U-box ligase with kinase domain, is targeted by the late blight pathogen effector PiSFI3, resulting in the inhibition of immune responses.¹⁹ Another potato U-box ligase, StPUB17, positively regulates plant immunity by degradation of the negative regulator StKH17.^20,21 Arabidopsis AtPUB25 and AtPUB26 negatively regulate plant immunity and disease resistance by degrading the inactivated immune kinase Botrytis-INDUCED KINASE 1 (BIK1).²² Conversely, AtPUB25 and AtPUB26 act as positive players conferring freezing tolerance by targeting the cold signaling negative regulator AtMYB15 for degradation.²³ Additionally, Arabidopsis AtPUB46,^24,25 rice OsPUB67²⁶ and wheat TaPUB1²⁷ are involved in promoting drought tolerance, whereas Arabidopsis AtPUB19,²⁸ AtPUB22 and AtPUB23,^29,30 and wheat TaPUB26³¹ serve as negative regulators in ABA dependent or independent abiotic stress responses.

The tea plant (Camellia sinensis) is an economically important woody crop that is extensively cultivated in tropical and subtropical areas including South China to produce the world’s most popular tea beverage.³² Tea plant throughout the entire life cycle is frequently challenged by diverse abiotic and biotic stresses, such as drought^33,34 and anthracnose pathogens,^35,36 resulting in serious quality and yield losses. However, the underlying molecular mechanisms on stress responses and resistance in tea plant remain poorly understood. Recently, several editions of the genomes of two varieties of tea plant, C. sinensis var. assamica (CSA)³⁷ and C. sinensis var. sinensis (CSS)^38–41 have been released successively, providing references for functional genomics research on response and adaptation to environmental stresses in tea plant and other Camellia species. Here, we describe the genome-wide identification, evolutionary relationship and expression analysis of tea plant U-box genes (CsPUBs) in response to the anthracnose pathogen Colletotrichum fructicola and drought stresses based on one of the chromosome-scale reference genomes of CSS, hopefully providing insights into U-box genes function on stress responses and breeding for resistance.

Materials and methods

Plant materials

The cultivar “Suchazao” of CSS (CSS-SCZ) was maintained in the Camellia Germplasm Resources Center of Guangxi Forestry Research Institute (GFRI). Two-year-old plants were grown in a growth chamber at 28°C with a photoperiod of 14 h and 80% humidity and used for pathogen and drought treatments in the present study.

Anthracnose pathogen infection

The anthracnose pathogen, C. fructicola, was isolated from diseased leaves of CSS-SCZ. The healthy and unwounded leaves (first-third leaves) were inoculated with conidial suspensions of C. fructicola (10⁶ spores/mL) as described previously.³⁵ The leaf tissues were sampled at 0 h (before inoculation), 24 h, 48 h and 72 h after inoculation (hai), immediately frozen in liquid nitrogen and stored at −80°C for RNA isolation.

Drought treatment

For drought stress, plants were treated by gradually withholding water for 14 d and then re-watering for 3 d. The leaf tissues (first-third leaves) were sampled at the withholding point 0 d, 3 d, 7 d, 14 d and after rewatering for 3 d, respectively.

Sequence retrieval and identification of CsPUB proteins

One edition of the chromosome-scale reference genome of CSS was used here⁴⁰ and downloaded from Tea Plant Information Achieve (TPIA, http://tpia.teaplant.org/).⁴² To identify the CsPUB genes, the Hidden Markov Model (HMM) profile for U-box domains (PF04564) were downloaded from Pfam database (http://pfam.xfam.org/).⁴³ HMM search in HMMER 3.3.2 (http://hmmer.org/download.html) against the CSS genome was performed with an expected value (e-value) threshold of <0.001. Meanwhile, protein blast against CSS genome database on TPIA with Arabidopsis PUB protein sequences downloaded from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/) was performed (e-value = 0.001). Taken these two results together, the final members of CsPUBs were obtained and confirmed by PfamScan (e-value = 0.001, https://www.ebi.ac.uk/Tools/pfa/pfamscan/)⁴⁴ and SMART (e-value = 0.001, http://smart.embl-heidelberg.de/).⁴⁵ The basic information of CsPUBs including intron number, intron length, chromosome localization was determined based on the CSS genome database.⁴⁰ The subcellular localization of CsPUB proteins was predicted using the TargetP 2.0 server (https://services.healthtech.dtu.dk/service.php?TargetP).⁴⁶

Classification and phylogenetic analysis of PUB genes

The conserved domains or motifs of CSS and Arabidopsis PUB proteins were identified using the online tools PfamScan (e-value = 0.001)⁴⁴ and SMART (e-value = 0.001).⁴⁵ According to the extra domain organization, the classification of CsPUB proteins was determined. Meanwhile, the phylogenetic trees were constructed by MEGA7 (www.megasoftware.net) based on Neighbor-Joining method with 1000 bootstrap replications and partial deletion.⁴⁷ A motif-based sequence analysis tool, the MEME suite tool (http://meme-suite.org/),⁴⁸ was used to identify extra conserved motifs in Class V PUB proteins. Gene duplication events of U-box genes were analyzed by MCScanX (http://chibba.pgml.uga.edu/mcscan2/)⁴⁹ and visualized by Cirsco software (http://www.circos.ca/software/).⁵⁰

Stress-related RNA-Seq data analysis

Two previously released stress-related RNA-seq data-sets of tea plant^35,51 were used in the present study to analyze the expression of CSS U-box genes. Expression levels of tea plant genes response to the anthracnose pathogen C. fructicola (accession numbers: SRX3142216, SRX3142220, SRX3142221, SRX3142226, SRX3142227, SRX3142228, SRX3142230, SRX3142231, SRX3142232)³⁵ and drought (accession numbers: SRX2234716, SRX2234790, SRX2234791, SRX2234821, SRX2234822, SRX2234823)⁵¹ were available from NCBI SRA (https://www.ncbi.nlm.nih.gov/sra). These two RNA-Seq data were remapped back to the CSS genome used here.⁴⁰ The differentially expressed genes (DEGs) were defined under the criteria of fold change| (FC) ≥ 1.5. Heatmaps and the Venn diagram were constructed using Excel and PowerPoint of Office 365, respectively.

Quantitative real-time PCR assays

Total RNAs were extracted from the stress-treated samples described above using TRIzol kit (Invitrogen, http://www.invitrogen.com). Quantitative real-time PCR (qRT-PCR) assays were performed as described previously³⁵ using an ABI PRISM 7500 Real-time PCR System (Applied Biosystems) and CsPUB specific primers (Table S1) following instructions of the supplier. CsGAPDH1 (CSS0032053) was served as an internal control.

Cis-acting elements analysis

The 1500 bp promoter sequences upstream of the ATG start codon of CsPUB genes were retrieved from TPIA.⁴² The cis-acting regulatory elements of CsPUB genes were predicted using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).⁵²

Results

Genome-wide identification of PUB genes in tea plant

To retrieve the PUBs genes from the tea plant, one of the chromosome level reference genome sequences of CSS⁴⁰ was used in the present study. The hidden Markov model (HMM) profile for U-box domain (PF04564) was obtained from Pfam database.⁴³ HMM search against the CSS genome was performed, and 86 PUB genes were identified from the tea plant. Furthermore, protein blast against the CSS genome database on TPIA⁴² with Arabidopsis PUB protein sequences was performed, and three more CsPUBs were identified. Overall, there are 75 CsPUBs located in all 15 chromosomes, and other 14 CsPUBs found in Contigs. According to the position on the chromosomes, these PUBs of tea plant were named as CsPUB1 to CsPUB89 in order. The basic information of AtPUBs and CsPUBs including the gene name, chromosome position, the number and average length of intron and protein length were listed in Table S2 and Table S3, respectively. Sixty-seven of 89 CsPUB genes in tea plant contained more than one intron, and the intron length was sharply divergent among the tea plant PUB gene family. The average intron length of CsPUBs was 948 bp, which is much larger than that of AtPUBs. The average length of introns of 17 CsPUB genes was larger than 1kb, whereas the average length of introns of all Arabidopsis PUB genes was shorter than 300 bp (Figure S1). Meanwhile, the length of CsPUB protein sequences ranged from 90 to 1066 amino acids, and the average protein length between CsPUBs and AtPUBs was of no significant difference in spite of the large divergence on intron length. Additionally, the PUB proteins of tea plant were predicted to be localized throughout the cell, including nuclear, cytoplasmic, plasma membrane, mitochondrial, chloroplast, and Golgi locations, and the majority of CsPUBs contained putative nuclear localization signal (Table S3).

Classification and phylogenetic analysis of the tea plant PUB proteins

The protein sequences of CsPUBs were used to search with PfamScan⁴⁴ and SMART⁴⁵ tools to identify the extra domains or motifs. The PUB proteins of Arabidopsis were analyzed and reconfirmed in the current study as well. As AtPUB62 (At3g49065) was removed by TAIR (https://www.arabidopsis.org/), 63 AtPUBs were introduced here. Various other domains/motifs were detected in PUB proteins of tea plant and Arabidopsis in addition to U-box domain. According to the presence of the extra domains/motifs, tea plant PUB proteins were grouped into ten classes, following and incorporating the previous defined classes in Arabidopsis (Table 1). The phylogenetic tree constructed with Arabidopsis and CSS PUB proteins supported the classification (Figure 1).

Table 1. Domain organizations of PUB proteins of tea plant and Arabidopsis.

Class	Domain organization (From N to C end)	Numbers
		CSS	Arabidopsis
I	UFD2 + U-box	1	1
II	U-box + ARM	45	27
III	U-box + PPIase	1	1
IV	Kinase + U-box	11	14
V	U-box only	23	15
VI	U-box + WD40	3	2
VII	TPR + U-box	2	1
VIII	MIF4G + U-box	0	2
IX	U-box + KAP	2	0
X	PRR + U-box	1	0
Total		89	63

Figure 1. Phylogenetic analysis of the PUB proteins from Arabidopsis and tea plant. The tree was constructed by MEGA7 based on Neighbor-Joining method with 1000 bootstrap replications and partial deletion.⁴⁷ The PUB proteins of Arabidopsis (AtPUBs in red circles) and CSS (CsPUBs in blue triangles) were classified into ten classes represented in different colors.

Similar to Arabidopsis, Class I consists of a single PUB protein, CsUFD2 (CsPUB15), which shares 23, 74% and 61% sequence identities with yeast UFD2, Arabidopsis AtUFD2 (AtPUB1) and rice OsUFD2, respectively. Class II with 45 CsPUBs and 28 AtPUBs containing the armadillo (ARM) domain was the largest class in CSS and Arabidopsis. The ARM repeat domain with approximately 40 amino acids was initially identified in Drosophila melanogaster⁵³ and found in all eukaryotes.⁵⁴ It was involved in protein–protein interaction and functioned in diverse cellular processes including self-incompatibility, hormone signaling and immunity in higher plants.^54–56

Class III was originally defined as the second largest class in Arabidopsis and rice PUB families.¹⁴ Recent analysis based on newly updated databases^44,45 showed that class III was composed of the PUB proteins with cyclophilin-type peptidyl-prolyl isomerase (PPIase) domain. Our analysis was consistent with the results described previously in barley¹³ and other plants.^57,58

The exact second largest class was Class V with 25 CsPUBs and 15 AtPUBs termed by containing U-box only without any other well-known or defined domains/motifs. To get more information about the structure of Class V PUB proteins, the motif structure of these 38 PUB proteins was analyzed. In addition to the U-box domain, seven extra undefined conserved motifs were identified (Figure 2). According to the motif organization, the Class V PUBs were categorized into five subclasses (Va – Ve). Class Va, Class Vb and Class Vd contained 3 to 5 extra motifs, while only one or none extra motifs were found in Class Vc. Two extra motifs, motif 6 and motif 7, specially existed in Class Ve. It may provide positive references for the functional study of these Class V U-box genes.

Figure 2. Motif structures and phylogenetic tree of Class V PUB proteins of Arabidopsis and tea plant. Seven extra conserved unknown motifs were identified from the Class V PUB proteins of Arabidopsis and CSS, which hence were further categorized into five subclasses according to the motif structures.

The third largest class, Class IV with 11 CsPUBs and 14 AtPUBs, harbored a kinase domain at the N-terminal region. Phosphorylation by protein kinases was another major type of PTM that controls diverse signaling pathways in eukaryotic cells.⁵⁹ This class PUB proteins with putative functions of both ligase and kinase might play significant roles in multiple cellular processes in higher plants.

Besides the classes mentioned above, there were several other smaller classes, including Class VI with WD40 repeats domain and Class VII with tetratrico peptide repeat (TPR) domain (Table 1). These two domains were documented to be involved in protein interactions.^60,61 WD40 repeats containing PUB proteins, CsPUB34, CsPUB46, AtPUB59 and AtPUB60, were homologous to yeast and human Prp19, which was involved in pre-mRNA splicing and DNA repair.^62,63 Additionally, it existed another CsPUB with WD40 domain, CsPUB73, which was clustered with Class II PUB proteins.

Moreover, similar to rice and barley, there was no MIFG domain containing PUBs found in CSS. Conversely, there were two smaller classes, Class IX with KAP domain and Class X with PPR repeat domain, identified only in CSS compared to Arabidopsis. KAP (kinesin-associated protein) domain was associated with motor function,⁶⁴ and PPR (Pentatricopeptide repeat) domain was involved in organelle biogenesis and mRNA processing.^65,66

Gene duplication analysis

Gene duplication events are of significant importance for gene family expansion. Here, the duplication events of the PUB genes in CSS genome were analyzed. There were two pairs of tandem repeats, CsPUB10/11 and CsPUB81/82, and 17 pairs of segmental genes between CSS chromosomes (Figure 3, red lines; Table S4). These results showed that both tandem and segmental gene duplications contributed to the expansion of the PUB gene family in CSS. Specially, it was indicated that the segmental duplication was the major driving force of the CsPUB gene expansion.

Figure 3. Duplication analysis of PUB genes of Arabidopsis and tea plant. The PUB genes in 5 chromosomes of Arabidopsis (in orange) and 15 chromosomes of CSS (in cyan) were introduced here. The orthologous pairs between AtPUBs and CsPUBs were highlighted in blue. The gene pairs of AtPUBs were colored in yellow, while the gene pairs of CsPUBs were colored in red. The gray lines marked the rest genes pairs in Arabidopsis and tea plant.

Furthermore, the collinear relationship between CSS and A. thaliana was completed (Figure 3, blue lines; Table S4). There were 49 orthologous gene pairs between CsPUBs and AtPUBs, of which 12 AtPUBs had two orthologous copies in CSS. It was implied that these orthologous genes might share similar function in these two species.

Expression analysis of CsPUB genes under anthracnose pathogen and drought stresses

Tea plant is frequently challenged by various environmental stresses, including anthracnose pathogens and drought. In order to investigate the expression patterns of CsPUBs under these two stresses, two recently published stress-related RNA-seq libraries were used in the current study. There were 14 and 28 CsPUB genes differentially expressed in response to anthracnose pathogen (Figure 4(a) and Table S5) and drought (Figure 4(b) and Table S6), respectively. Among them, eight CsPUB genes shared overlapping roles responding to both stresses (Figure 4(c)). Furthermore, six of the eight CsPUBs, CsPUB1, CsPUB27, CsPUB33, CsPUB40, CsPUB73 and CsPUB88, were significantly upregulated under these two stresses. The expression levels of these six CsPUBs under pathogen infection and drought treatment were confirmed by qRT-PCR (Figure 5), which were consistent with the result of RNA-seq analysis.

Figure 4. Expression profiles of CsPUB genes under C. fructicola infection and drought treatment. (a) 14 CsPUB genes were differentially expressed responding to anthracnose pathogen; (b) 28 CsPUB genes were differentially expressed responding to drought stress; (c) Venn diagram analysis of the 34 total differentially expressed CsPUBs under these two stresses. Eight CsPUBs were expressed differentially under both stresses, while six of the eight CsPUBs (in bold) were upregulated under both stresses.

Figure 5. Expression patterns of the six CsPUB genes confirmed by qRT-PCR. (a) Expression levels of the six upregulated CsPUBs under C. fructicola attack; (b) Expression levels of the six upregulated CsPUBs under drought treatment. Three independent experiments were performed. CsGAPDH1 acted as the internal control gene to normalize the expression data.

Stress-associated cis-acting elements in the six CsPUB promoters

To get more evidences for the six upregulated CsPUB genes on stress responses, the cis-acting regulatory elements in the promoters of the six CsPUBs were predicted by PlantCARE.⁵² There were 11 well-known stress-related elements identified (Figure 6). STRE (stress response element; AGGGG) was involved in rapid response to abiotic stresses such as heat and osmotic stresses,^67,68 while the ARE (antioxidant response element; AAACCA) was related to the anaerobic and oxidative stress induction.⁶⁹ WUN-motif (wound-responsive element; AAATTACT), TC-rich repeats (ATTCTCTAAC),⁷⁰ and G-box (CACGTG)⁷¹ were three types of elements involved in biotic stress response and defense. ABRE (abscisic acid response element; ACGTG) and ERE (ethylene response elements; ATTTTAAA) were related to the major stress-related hormones ABA and ethylene, respectively. W box (WRKY binding site; TTGACC), MBS (MYB binding site; CAACTG), MYB (TAACCA) and MYC (CATTTG) were associated with the transcription factors WRKY, MYB and MYC, respectively, which were involved in environmental stresses response and adaptation.^72,73 These results further provided auxiliary evidence for the six up-regulated CsPUB genes in response to abiotic and biotic stresses.

Figure 6. Stress-related cis-acting elements in the six upregulated CsPUB genes. The cis-acting regulatory elements in the promoters of the six CsPUBs were predicted by PlantCARE.⁵² Eleven well-known stress-related elements were identified, which were reported to be involved in environmental stress response and adaptation. The degree of red represents the number of the elements in the CsPUB promoters.

Discussion

The PUB gene family, one of the largest ubiquitin ligase families in plants, was known to play significant roles in plant response to biotic and abiotic stresses. More and more evidences were reported about the functional characterization of PUBs in Arabidopsis,^22,23 rice¹⁸ and other plants.^19–21,31 However, the function of tea plant PUB genes on stress response is largely unknown. Here, we identified 89 PUB genes from tea plant genome. There were 14 pathogen-induced CsPUBs and 28 drought-induced CsPUBs identified from two recently published stress-related RNA-seq libraries, respectively. Among them, the expression levels of six CsPUB genes were significantly upregulated in response to both anthracnose pathogen and drought stresses.

The CsPUB and AtPUB proteins were categorized into ten classes based on the domain organization, which was consistent with the adjusted structure of U-box genes described by Ryu et al.¹³ There were two CSS special classes of PUB genes identified from tea plant, Class IX with KAP domain (CsPUB35 and CsPUB54) and Class X with PRR repeats (CsPUB85). KAP domain was shown to be involved in motor function⁶⁴ and was found in U-box proteins of Medicago truncatula as well.⁵⁷ PPR domain was reported to be associated with organelle biogenesis and mRNA processing.^65,66 Moreover, these three CsPUBs were closely neighbored to the Class II PUB proteins with ARM domain. Hence, the functional characterization of these three CsPUB genes is needed. Compared to Arabidopsis PUB gene family, the CsPUB family was with a notable expansion in total. Obviously, the larger Class II of CsPUBs in tea plant mainly led to the expansion, over a half of CsPUBs belonged to Class II. Furthermore, it was indicated that the tandem and segmental gene duplications contributed to the CsPUB family expansion. Additionally, the number of Class V CsPUB genes also slightly increased in comparison to that of Class V AtPUBs. Class V was ever defined as the U-box proteins without known domains. However, we identified several undefined or unknown conserved motifs from Class V PUB proteins in Arabidopsis and tea plant. The motifs structure of Class V varied widely, and it could be further assorted into five different subclasses. It might be positively helpful for the further investigation of Class V U-box genes.

Based on previously released stress-related RNA-seq data, we identified approximately 40% of CsPUB genes expressed differentially responding to drought or fungal attack by C. fructicola. Among them, eight CsPUB genes were differentially expressed in response to both anthracnose pathogen and drought stresses. Particularly, six of the eight CsPUBs were up-regulated under these two stresses. CsPUB1, CsPUB33 and CsPUB88 all belonged to Class II. CsPUB73, though with WD40 domain, was also clustered with Class II CsPUB proteins with ARM domain. Multiple evidences showed that ARM domain containing proteins were involved in diverse cellular processes including cytoskeletal regulation, flowering, self-incompatibility and disease resistance.^54–56 Specially, the closest ortholog of CsPUB33 and CsPUB88, Arabidopsis AtPUB16 and AtPUB17, were reported to function in plant defense.^74,75 CsPUB27, the Class Vd U-box protein with undefined conserved motifs, shared high sequence similarity to its closest ortholog AtPUB27, which was immediately activated on infection with a pathogen-derived elicitor.⁷⁶ CsPUB40 was the only Class IV U-box gene among the six upregulated genes. Class IV CsPUBs harbor a kinase domain with putative kinase function, which play central roles in multiple cellular processes through its phosphorylation.⁵⁹ The function of this Class U-box genes in Arabidopsis and other plants remains largely unknown. Referentially, a well-characterized gene encoding a protein with both E3 ligase and kinase activities, namely KEEP ON GOING (KEG), plays critical roles in seedling establishment,⁷⁷ hormone signaling^77–79 and stress responses.^80,81 These evidences implied that the six CsPUB genes might putatively function on stress responses. However, whether and how these CsPUB genes play dual positive roles in response to both biotic and abiotic stresses, more proofs are required.

In conclusion, there were 89 U-box genes identified from tea plant, while six of them were upregulated under both anthracnose pathogen and drought stresses. Further investigations on these six CsPUB genes are needed to clarify the potential positive roles and underlying molecular mechanisms in biotic and abiotic stress responses.

Author contributions

J. L. M., R. Q. Z. and H. Z. L. conceived and designed the experiments; H. Z. L., W. J. L. and D. X. Z. performed the experiments and analyzed the data; H.Z.L., R. Q. Z. and J. L. M. wrote the paper.

Acknowledgments

We thank Dr Hang Ye and Ms Yu Wei from GFRI for their assistance in this study. The sweet wife (Y. Y.) and the lovely son (C. Y. L.) of H. Z. L. are acknowledged for their warm support.

Disclosure statement

The authors declare no conflict of interests.

Supplementary Material

Supplemental data for this article can be accessed on the publisher’s website.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by the grants from Guangxi Key Laboratory of Special Non-wood Forest Cultivation and Utilization (JA-20-02-01) and Guangxi Natural Science Foundation (2020GXNSFAA297045).