Comprehensive identification and expression analysis of CAMTA gene family in Phyllostachys edulis under abiotic stress

Identification of CAMTA genes in P. edulis genome

All files associated with the whole genome sequence data of Phyllostachys edulis were downloaded from the database website (http://gigadb.org/dataset/100498). A numerical tabular Hidden Markov Model (Profile HMM) was constructed using HMMER3 (http://hmmer.janelia.org/) to match the Phyllostachys edulis protein database (significant E value set to no more than 1 × 10⁻²⁰) (Finn, Clements & Eddy, 2011). The CAMTA domain (PF03859) obtained from the Pfam database was screened and integrated (Finn et al., 2016), and the candidate gene family members were obtained from the initial screening. The CAMTA structural domains of the candidate family members were analyzed using SMART (Letunic, Doerks & Bork, 2012), along with the Plant TFDB and NCBI BLAST for further comprehensive analysis and identification to obtain candidate CAMTA transcription factor families (Jin et al., 2017).

Physicochemical properties and signal peptide analysis of P. edulis CAMTA

The Sequence Toolkits module of TBtools software (v1.098765) was used to derive the coding sequence (CDS), protein fasta sequence, gene structure and gene location information of CAMTA gene family members from the corresponding genome-wide database (Chen et al., 2020) using The online tools Prot Param and TargetP 2.0 Server (https://services.healthtech.dtu.dk/service.php?TargetP-2.0) were used to analyze their physicochemical Properties and signal peptides were analyzed.

Phylogenetic analysis

The whole genome information of O. sativa, Arabidopsis, Z. mays, and Brachypodium distachyon was downloaded from the O. sativa genome database (https://shigen.nig.ac.jp/rice/oryzabase/), the Arabidopsis database (http://www.arabidopsis.org), Z. mays database (https://maizegdb.org/), and B. distachyon database (https://phytozome-next.jgi.doe.gov/info/Bdistachyon_v3_1), respectively, and based on the obtained CAMTA Protein sequences of the four plants, the software ClustalX2.1 was used to contrast the CAMTA Protein sequences of P. edulis. The sequence alignment results were used to construct phylogenetic trees by the software MEGA7 using the neighbor-joining (NJ) method, and the bootstrap evaluation (Bootstrap) was repeated 1,000 times.

Analysis of gene structure, motifs, domains

Based on the gene location information of P. edulis genome annotation file (GFF), the gene intron and exon sequences were analyzed and the gene structure of PeCAMTA gene family was visualized; the NCBI online software CDD was used to forecast the conserved structural domains of CAMTA gene family members, and their amino acid conserved sequences were predicted using the online software MEME (Bailey et al., 2009).

Cis-acting elements in the PeCAMTA gene promoter regions

PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to identify cis-acting elements in the 1,500 bp promoter region upstream of the transcription start site for each gene, and the results were submitted to TBtools (v1.098765) for visualization.

Chromosome distribution and interspecies covariance analysis

The BLAST module of TBtools (v1.098765) software was used to execution sequence comparison of all proteins in the genome of bamboo, and two-way alignment of P. edulis with O. sativa and P. edulis with Arabidopsis, based on genome-wide GFF files, using MC ScanX, Circos (0.69–9) and Multipe Synteny Plot. CAMTA gene family chromosome distribution and interspecies covariance were visualized using MC ScanX, Circos (0.69–9) and Multipe Synteny Plot.

Tissue-specific expression levels of PeCAMTA genes

In order to analyze the specific expression of CAMTA gene in P. edulis, we downloaded RNA-seq data from the NCBI gene expression profiles database (Accession: ERR105067–ERR105076). Transcriptome data which was quantified as transcripts per million reads (TPM) were log2-transformed (Cushion et al., 2018).

Plant material, RNA extraction and qRT-PCR analysis

Normal-grown 3-month-old live P. edulis seedlings were used as the control group with the following abiotic stress treatments: 4 °C, 42 °C and 500 ml 30% PEG6000 (The effect obtained by PEG6000 induced water shortage is the same as that obtained by progressive drought of the soil); sampled at 0, 3, 6, 12 and 24 h for the above treatments, and at 0, 3, 6 and 12 h for 42 °C-treated live P. edulis seedlings, and the second youngest leaf from top to bottom was snap-frozen in liquid nitrogen and saved in a −80 °C freezer.

Extraction of total RNA using an RNA extraction kit (Kangwei Century Biotechnology Co., Ltd., Taizhou, Jiangsu, China). cDNA was synthesized using a Script RT kit (Takara Bio, Kusatsu, Japan) kit and used for subsequent qRT-PCR assays. For the 11 identified PeCAMTA genes, qRT-PCR primers were designed online using Primer Premier 3, with P. edulis NTB (nucleotide tract-binding protein) as the internal reference gene (Fan et al., 2013). SYBR qPCR Master Mix (Code. Q311-02, Nanjing, China) was used to perform qRT-PCR in Multiplate™ 96-well PCR plates (Bio-Rad, Hercules, CA, USA). Each sample was tested using three technical replicates to ensure the accuracy of results. The reaction conditions refer to the method of Ma R (Ma et al., 2021).

Statistical analysis was conducted with an analysis of variance (ANOVA) and Independent-Sample T Test using Statistical Package for the Social Sciences (SPSS) software (version 24.0; IBM SPSS Statistics, Armonk, NY, USA).

Identification and characterization of PeCAMTA genes in P. edulis

Candidate family members were searched by the plant CAMTA Pfam (PF04770) model, and a significant E value of no more than 1 × 10⁻²⁰ was set for preliminary screening. A total of 11 CAMTA gene family members were obtained by combining gene structure, chromosomal localization, conserved structural domains and other characteristics, and removing gene duplicate transcripts and non-full-length amino acid sequences. As shown in Table 1, the CAMTA gene family genes were renamed PeCAMTA01 to PeCAMTA11 based on the chromosomal positioning information of the genes. bioinformatics analysis of the protein sequences of the 11 family members showed that the largest protein molecular weight of the CAMTA gene family members was 114.92 kD, and the smallest protein molecular weight was 90.10 kD. The amino acid sequence lengths ranged from 816 to 1,031 aa. The isoelectric points lie between 5.18 and 8.2. Two of the family proteins are basic (theoretical isoelectric point > 7) and nine are acidic (theoretical isoelectric point < 7). The aliphatic amino acid index revealed that the thermal stability of the proteins of this family was between 74.03 and 80.82, suggesting that the proteins of this family have small differences in thermal stability. Signal peptide analysis showed that none of the 11 members had signal peptides, indicating that the protein sequences of the CAMTA genes of P. edulis do not have transmembrane structures.

Phylogenetic analysis

Phylogenetic analysis of amino acid sequences of P. edulis CAMTA, O. sativa CAMTA, Arabidopsis CAMTA, Z. mays CAMTA and B. distachyon CAMTA was performed, and we classified the amino acid sequences of PeCAMTAs into five subbranches (I~V) (Fig. 1) according to Wang et al. (2022) classification method, among which the protein sequences of Arabidopsis CAMTA genes were classified into one subclade, and the amino acid sequences of O. sativa, Z. mays and P. edulis CAMTA genes were grouped into one subclade. It is more closely related to O. sativa and Z. mays, and more distantly related to Arabidopsis.

Gene structure, conserved domains, motifs and sequence analysis

Analysis of the gene structure of PeCAMTA gene family showed that the number of introns (intron) of each PeCAMTA gene ranged from 10 to 14. The 11 sequences were divided into four categories, because the affinities of P. edulis in other species make the results differ from the classification in the evolutionary tree. Gene PeCAMTA09 in subfamily III contains the longest intron region, while gene PeCAMTA04 in subfamily II and gene PeCAMTA11 in subfamily III have the shortest introns.

PeCAMTA gene family was further analyzed for conserved structural domains based on the NCBI online software CDD, as shown in Fig. 2C. As shown, all CAMTA gene family members contained CG-1 structural domains located at the N terminus, PeCAMTA10, PeCAMTA05, PeCAMTA03 in the first subclade and PeCAMTA11, PeCAMTA06 in the third subclade had TIG structural domains in addition to the typical CG-1 structural domains, while PeCAMTA08 in the first subgroup and PeCAMTA09 in the third subgroup do not have TIG structural domains, and both the second and fourth subgroups contain both CG-1 and TIG structural domains. All of the first subgroup contained ANKYR structural domains, PeCAMTA07 in the second subgroup and PeCAMTA09 and PeCAMTA06 in the third subgroup contained ANKYR structural domains, and the rest of PeCAMTA01 and PeCAMTA04 in the second subgroup, PeCAMTA11 in the third subgroup and the fourth subgroup did not contain ANKYR structural domains. All of the second subclade contained IQ structural domains, PeCAMTA08, PeCAMTA10, PeCAMTA03 in the first subclade, PeCAMTA11, PeCAMTA09 in the third subclade and the fourth subclade contained IQ structural domains, and the remaining PeCAMTA05 in the first subclade and PeCAMTA06 in the third subclade did not have IQ structural domains. PeCAMTA08, PeCAMTA10, PeCAMTA03 in the first subfamily and PeCAMTA07 in the second subfamily have all CAMTA structural domains.

The members of the P. edulis CAMTA gene subgroup contain motifs numbering 6 and 8, which are highly conserved, of which motif 1, motif 6 and motif 7 constitute the CG-1 structural domain. Except for PeCAMTA05, which lacks motif 7 and motif 3 in the first family, the genes in the other families have all motifs.

Chromosomal location and gene duplication of PeCAMTA genes

The chromosome distribution (Fig. 3) of the PeCAMTA gene family showed that 11 PeCAMTA genes were distributed on nine chromosomes with different densities of chromosomal gene distribution. Based on intraspecific covariance analysis (Fig. 4A), only the genes PeCAMTA08 and PeCAMTA10 were found to have undergone gene duplication (tandem duplication), while the remaining genes did not show gene duplication. The results indicated that only individual genes caused amplification of CAMTA transcription factor members on different chromosomes through gene duplication.

As shown in Fig. 4, only three P. edulis CAMTA homologous protein genes occur in Arabidopsis chromosomes, while eight P. edulis CAMTA genes can be found on six Z. mays chromosomes with corresponding paralogous homologs, and nine P. edulis CAMTA genes can be found on five O. sativa chromosomes with corresponding paralogous the same genes were found on five O. sativa chromosomes. Therefore, the covariance between P. edulis and O. sativa and Z. mays was more significant than that between P. edulis and Arabidopsis. In addition, most of the genes in the O. sativa and Z. mays CAMTA families have more than two paralogous homologs in P. edulis, inferring that there may have been a massive gene doubling event in the P. edulis CAMTA gene family in the evolution process.

Cis-element analysis of PeCAMTAs

The P. edulis CAMTA gene family members contain 11 genes extracted upstream to 1,500 bp nucleotide sequences, and promoter prediction revealed that in addition to the core promoter elements, many other cis-acting elements were found (Fig. 5), such as light-responsive elements, hormone-response-related elements and stress-responsive elements related to plant growth and development. The most abundant were hormone response-related elements, with all gene promoters containing at least one light response element and most gene promoters containing at least one phytohormone response element. The stress response elements include low temperature stress response components, drought stress response components, anaerobic induction response components and other abiotic stress response components. All PeCAMTAs contained the drought stress response component MYC, and the drought stress response component MYB was the most abundant response element, suggesting that PeCAMTAs plays an essential role in drought stress response. The results suggest that different components of the promoter region of P. edulis CAMTA gene family may be important in regulating plant growth and development and in resisting abiotic stresses.

Tissue-specific expression levels of PeCAMTA genes

To study expression of CAMTAs family genes (transcriptome) in different organs of P. edulis (Fig. 6). The expression levels of CAMTAs in four tissues (leaf, stem, whip and root) were assessed by RNA-seq data. Gene expression profiles in different tissues indicated that CAMTA has different functions in P. edulis. The results showed that PeCAMTA07 and PeCAMTA11 expression profile was higher than other genes. Except for PeCAMTA11, the expression of the other PeCAMTA genes was higher in leaves than in stems, whips and roots. Moreover, PeCAMTA07 was more highly expressed in each tissue, indicating that this gene plays an important role in the overall development of P. edulis.

Expression profiles of the PeCAMTA genes during abiotic stress

To investigate the expression of PeCAMTAs during abiotic stress, we analyzed the expression of 11 PeCAMTAs under three abiotic stresses using qRT-PCR: polyethylene glycol (PEG), heat, and cold treatment. The expression patterns of PeCAMTAs responded differently to the three abiotic stresses, and some PeCAMTAs were either significantly induced or repressed. The expression pattern of most genes changed significantly during the early phase (0–6 h) of the stress response.

Under cold stress (Fig. 7A), expression peaked at 6 h for all genes except PeCAMTA02, with a trend of up-regulation followed by down-regulation, with PeCAMTA02 peaking at 3 h followed by down-regulation. PeCAMTA03 and PeCAMTA06 expression peaked higher than the other genes, at about 6. PeCAMTA01, PeCAMTA02, PeCAMTA03, PeCAMTA05, PeCAMTA07, PeCAMTA08, PeCAMTA10 and PeCAMTA11 expression was below about 0.3 at 12 h and tended to be 0 at 24 h. PeCAMTA04, PeCAMTA06, PeCAMTA08, PeCAMTA09 and PeCAMTA10 expression at 3 h was significantly lower than in the untreated condition (CK).

Under heat stress (Fig. 7B), all genes reached peak expression at 6 h, with higher peaks at 22.4 for PeCAMTA03 and 24 for PeCAMTA11, and the expression trends were all up-regulated and then down-regulated. All genes were expressed at lower levels at 3 h than untreated (CK), with PeCAMTA04, PeCAMTA06, PeCAMTA07, PeCAMTA08, PeCAMTA09 and PeCAMTA11 all converging to 0 at 3 h.

Under drought stress (Fig. 7C), all genes reached peak expression at 6 h, with the highest peak being 34 of PeCAMTA11. The expression trend was mostly up-regulated, then down-regulated and finally up-regulated, with only PeCAMTA06 and PeCAMTA10 having lower expression at 24 h than at 12 h. All genes except PeCAMTA01 had no significant difference in expression from the control (CK) at 3 h.

To better compare the differences in expression of the PeCAMTA gene family under the three stresses, the expression values of each gene under different stresses were compared together (Fig. 8). The comparison showed that, except for a few cases (e.g., PeCAMTA11, where heat stress expression was higher than drought stress at 12 h), the majority of cases were higher for drought stress than for heat and cold stress, whether at 3, 6, 12 or 24 h. In addition, drought stress expression was up-regulated at 24 h.

To compare expression differences between different genes, the expression of all genes under the same stress was compared (Fig. 8). Under cold stress, PeCAMTA11 had higher expression at 3 h and PeCAMTA03 at 6 h, while PeCAMTA09 had higher expression at both 3 and 6 h. Under drought stress, PeCAMTA08 had lower expression at all stages, PeCAMTA11 had higher expression at 6 h and was in an average position at both 12 and 24 h. Under heat stress, PeCAMTA03 and PeCAMTA11 had higher expression at 6 h and PeCAMTA11 also had higher expression at 12 h.

Genome-wide identification and phylogenetic analysis of the PeCAMTA gene family

CAMTAs are a specific class of plant transcription factors that play an essential role in the regulation of plant growth and development and metabolism (Galon et al., 2008; Yang et al., 2012; Yang & Poovaiah, 2002). The molecular functions of CAMTA have been verified not only in Arabidopsis (Galon et al., 2010; Pandey et al., 2013) and O. sativa (Gain et al., 2022) as model plants, but also in cotton (Pant et al., 2018), maize (Yue et al., 2015), tobacco (Kakar et al., 2018) and tomato (Yang et al., 2012), where the CAMTA gene family has been gradually identified. However, no studies on CAMTAs have been conducted in the economically important bamboo species, P. edulis. Currently, the draft genome of P. edulis is largely complete, allowing for a full identification of key gene families (Peng et al., 2013; Zhao et al., 2018). We classified the 11 PeCAMTA genes into five categories, Class I, Class II, Class III and Class IV, based on phylogenetic analysis. Among them, four members (36%) belonged to Class I, three members (27%) to Class II, three members (27%) to Class III, and one member (9%) to Class IV (Fig. 1).

Gene structure analysis reveals structural differences among members within the same subfamily. Such as, PeCAMTAs members in the same family I have intron numbers ranging from 10–14. Therefore, we hypothesize that members of subfamily I may have undergone pruning of gene fragments during their evolution (Li et al., 2016; Staiger & Brown, 2013). Nevertheless, the similar conserved sequences and gene structures among CAMTA gene family members suggest that gene biological functions are generally the same within a family. All six NTR1 homologs of Arabidopsis have a conserved structural feature with a DNA-binding region (CGCG structural domain) at the N-terminal end and a CaM-binding structural domain at the C-terminal end. The role of Ca²⁺/CaM may be expressed in controlling interactions with other proteins or altering transcriptional activation of other proteins. In addition, conserved domain comparison showed that all PeCAMTA genes have CG-1 structural domains, indicating that the conserved motifs of the CAMTAs family are broadly conserved during evolution.

During signal transduction, multiple cis-acting elements on a gene promoter work together to regulate multiple complex biological responses. Solanum lycopersicum CAMTA gene contains salt stress regulatory elements, including ABRE, G-box, MBS, and TGA (Wang et al., 2021), and CAMTAs of different species have been reported to respond to a variety of biotic and abiotic stresses, including low temperature, hormones, high salt, and drought. Two genes, ZmCAMTA4 and ZmCAMTA6, were highly expressed under stress treatment, and cis-element analysis revealed the involvement of CAMTA genes in the association between environmental stress and stress-related hormones, and the GhCAMTA gene family may also be involved in the phytohormone signaling pathway (Liu, Hewei & Min, 2021; Pant et al., 2018). On the basis of the PlantCARE software, we found that elements involved in abscisic acid response, MeJA response, growth hormone (IAA) and many other hormone regulation-related elements were present. Therefore, we suggest that PeCAMTA genes may also be involved in the stress response of plants. Interestingly, the promoter regions of most PeCAMTA genes have the largest number of MYB elements involved in drought induction (Fig. 5). Recent studies on wheat confirmed that the expression of TaCAMTA1a-B and TaCAMTA1b-B. A total of 1 was down- and up-regulated, respectively, in response to drought stress to maintain normal physiological functions associated with the plant, and wheat CAMTA gene family members also contain a large number of MYB elements (Wang et al., 2022). The previous study also gave us ideas for subsequent stress experiments, and it became important to verify the expression of PeCAMTAs in response to drought stress.

Evolutionary characterization of the PeCAMTA gene family

Gene duplication may produce new genes, which greatly helps in the evolution of gene function. The three evolutionary patterns of gene replication are (Liu et al., 2019): segmental duplication, tandem duplication and translocation events. Segmental and tandem replication are the most common basis for gene family expansion in plants (Freeling, 2009; Li & Barker, 2020). Previous studies on whole genome duplication have shown that the genome size of bamboo (2,051.7 Mb) is similar to that of its close relative Z. mays (2,066.4 Mb), but the number of CAMTA genes is higher in bamboo than in maize (Chen et al., 2020). Therefore, we performed a consistency analysis within and among the P. edulis genomes. Within the P. edulis genome, there was one pair of segmental duplication genes in the CAMTA gene. Therefore, the amplification of the CAMTA gene family mainly comes from gene fragment replication. Simultaneous analysis of the genome of Bamboo and three other sequenced plant genomes showed that the members of the bamboo CAMTA gene family had significant consistency with the genomes of the monocot plant O. sativa.

The role of PeCAMTA genes in different tissues and organs

Several studies have shown that CAMTAs can regulate plants during lateral organ development and have an important effect on plant organ formation (Rahman et al., 2016; Shangguan et al., 2014; Wang et al., 2015; Yang et al., 2015), which is consistent with our findings. Analysis of expression profiles in different tissues of bamboo revealed that a large number of PeCAMTAs showed the amount of expression varies in different tissues (Fig. 6). PeCAMTA07 was up-regulated in all P. edulis growth sites, with expression in leaves, whips and roots more than twice that of other genes, indicating its importance in P. edulis growth and development. PeCAMTA11 was most abundantly expressed in flowers, suggesting that PeCAMTA11 is involved in flower bud differentiation in P. edulis, but other PeCAMTAs were more abundantly expressed in leaves than in flowers, suggesting that they are mainly involved in the plant growth process, but not in the plant bud differentiation process.

Expression of PeCAMTA genes in responses to cold, drought and heat treatments

Studies have shown that regulatory elements in the promoter region are important for the regulation of the gene in different environments (Wang et al., 2023). The E₂F/DP gene in P. edulis is mainly involved in drought stress response, and the PheE₂F/DP promoter contains many MYB and MYC2 binding sites (Li et al., 2021). Many MYB and MYC2 promoters were also identified in the TaCAMTA gene family, and TaCAMTA plays an important role in the physiological activities of wheat in response to drought stress. Using real-time fluorescence quantification assays, the results of the PeCAMTAs promoter analysis were combined to verify whether PeCAMTAs is involved in drought stress regulation. Comparison with other stresses showed that the expression of the PeCAMTA gene family was significantly higher under drought stress than under heat and cold stress, with higher expression at 6, 12 and 24 h. The expression of PeCAMTA11 was superior under all three stresses, which could be used as a reserve for the next gene function validation assay. Our study suggests that the PeCAMTA gene family plays a crucial role in drought stress response, but further studies are needed to elucidate the functional significance of the CAMTA gene family in P. edulis.