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. 2025 May 30;25:731. doi: 10.1186/s12870-025-06783-5

Comprehensive identification and expression analysis of the CPP gene family in maize (Zea mays L.)

Lei Gu 1,2,#, Tianyu Kang 1,#, Tuo Zeng 1,2, Hongcheng Wang 1, Bin Zhu 1, Xuye Du 1, Yinglang Liu 1,2,✉
PMCID: PMC12124014  PMID: 40442593

Abstract

Background

Maize is an important crop. The cysteine-rich polycomb-like protein (CPP) is crucial for plant development and abiotic stress response. However, few reports have been reported on the function of ZmCPPs. This study conducted bioinformatics and expression analyses of the ZmCPP gene family.

Results

Based on the homologous comparison methods, 12 ZmCPPs were identified in the last assembly of maize B73 genome (V5). They were unevenly distributed across six maize chromosomes. Six ZmCPPs formed 3 groups due to segmental but no tandem duplication, indicating segmental duplication as the key driving force of the ZmCPPs family expansion. Homologous evolutionary analysis classified 12 ZmCPPs into three groups, with each containing four members. ZmCPP gene structure and protein motif in the same group were highly conserved. The promoter regions of 12 ZmCPP genes containing plant growth, hormone, and abiotic stress-responsive elements. RNA-seq data indicated that the expression pattern of ZmCPPs exhibited organizational specificity and the ZmCPPs transcript levels could be influenced by abiotic/biotic stresses. RT-qPCR analysis of six ZmCPPs (ZmCPP2/4/8/9/11/12) showed that the expression of ZmCPPs were induced or reduced by short-term heat, cold, dehydration, and waterlogging stresses. Furthermore, ZmCPP2/9 was localized in the cytoplasm and nucleus, without transactivation activity in yeast.

Conclusion

Taken together, the comprehensive analysis of ZmCPPs in the whole genome provides a novel perspective on the evolutionary relationship among ZmCPP genes and lays a foundation for further study of the biological functions of ZmCPPs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06783-5.

Keywords: Maize, CPP gene family, Genome-wide analysis, Bioinformatics analysis, Abiotic stress

Background

Maize is an important crop worldwide. Severe abiotic stresses (e.g., extreme temperature, drought, and salinity) affect maize growth and development, posing a serious threat to important traits and resulting in notable decreases in yield [1]. After plants perceive stress signals, transcription factors (TFs) are responsible for transducing and amplifying the signals, thereby inducing the expression of related genes in cells to cope with the damage caused by abiotic stress [2]. Compare to the large plant TF families, such as MYC, MYB, NAC, WRKY, AP2/ERF; The cysteine-rich polycomb-like protein (CPP) transcription factor family with relatively few members, but it plays an important regulatory role in plant growth, development, and responses to abiotic stress [3]. The distinctive feature of CPP proteins is that their sequences contain two conserved CXC domains (motif: CXCX4CX3YCXCX6CX3CXCX2C, CXC1 and CXC2), as well as a short conserved sequence (RNPXAFXPK) for separated between these two CXC domains [4, 5]; These conserved region (CXCs and short linker) in CPP proteins is also known as the CRC/TCR /CHC domain, which an important characteristic of its family, responsible for binding to the promoter of the target genes and regulating its transcription [6, 7].

Based on the development of sequencing technology, the CPP gene family has been identified through bioinformatics analysis in many plants, such as Arabidopsis (8 genes) [8], rice (11 family members) [8], tomato (6 members) [4], Moso bamboo (Phyllostachys edulis) (17 genes) [5], soybean (20 family members) [9], wheat (37 genes) [10], and cucumber (Cucumis sativus) (5 members) [3]. The reported functions of the CPPs are primarily focused on their involvement in plant floral organ development and responses to abiotic environmental conditions; The TSO1/AtCPP5 gene was the first identified and characterized CPP member in plants through map-based cloning analysis [11]. The TSO1 act as an important regulatory protein in the mitosis of flower cells in Arabidopsis, however, this specific function seems to be not present in other tissues [12]. Knockout of the TSO1 gene in Arabidopsis disrupted the mitosis of floral organ cells, leading to a significant increase in DNA content within the nucleus; Additionally, the cell walls were incomplete at the end of mitosis, resulting in abnormal phloem development in the mutant lines, ultimately, these changes caused abnormal development of petals, stamens, and carpels [7, 13]. Further analysis indicated that TSO1 may through transcriptionally repress the expression of AtMYB3R1 gene to positively modulating the balance of shoot or root cell proliferation and differentiation [14]. In soybean, GmCPP1 may be repressing the expression of Gmlbc3 (leghemoglobin) to modulate the development of symbiotic root nodules [15].

Besides their function in tissue development, CPPs also respond to many environmental treatments (e.g., heat, drought, and salt). Soybean contain 20 CPPs, and most of them could induced by heat shock, implying its function in plant temperature stress response [9]. Tomato included 7 CPP genes, except for SlCPP5, the remaining six genes were all induced by drought treatment [4]; Further analysis revealed that tomato lines with gene silencing of SlCPP3 significantly reduced drought tolerance [4]. A recent study indicated ZmSK1 could negatively regulate drought tolerance [16]; Further analysis revealed ZmSK1 could phosphorylate ZmCPP2, resulting in ZmCPP2 losing the ability to directly upregulating the expression of ZmSOD4, causing a drought-sensitive phenotype in maize [16]. While the function of other ZmCPPs in maize nutritional growth and the abiotic stress response is largely unknown.

Previous reports revealed maize contains 13 ZmCPP genes based on B73 V3 genome sequence data [17]. In this study, 12 ZmCPP family members were identified in the latest assembled maize genome (V5, Zm-B73-REFERENCE-NAM-5.0) and characterized. We also explored the expression patterns of ZmCPPs under normal and abiotic (heat, cold, dehydration, and waterlogging) conditions. The subcellular localization and transcriptional activation activity of ZmCPP2 and ZmCPP9 were further analyzed. This work provides a comprehensive perspective on the ZmCPP gene members in maize and may be helpful for future research on understand the functions of ZmCPPs.

Results

Identification and characterization of ZmCPP genes

In this study, we used AtCPPs [8], OsCPP protein sequence [8] and the hidden Markov model (HMM) of the core CXC motifs (pfam03638) as detectors to homology comparison of the maize latest assembled genome (V5); After combining the blast results, eliminating the redundant transcripts and confirming the presence of complete CXC domain, 12 putative ZmCPPs were identified (Fig. 1); These 12 members were unevenly distributed across six chromosomes (the other four chromosomes not contained ZmCPP genes) (Fig. 1). Chromosome 1 and 3 both included the most ZmCPPs (three genes), followed by chromosomes 6 and 8 each contains two members, chromosomes 5 and 7 each only included one ZmCPP (Fig. 1). We named the 12 ZmCPPs as ZmCPP1 to ZmCPP12 basing on their relative positions in the chromosome (Fig. 1). The Open Reading Frame (ORF), molecular weights and amino acids (aa) of ZmCPPs ranged from 1071 bp/39.67 KDa/356aa (ZmCPP11) to 2403 bp/84.98 kDa/800aa (ZmCPP10) (Table 1). The isoelectric points (PI) of all ZmCPPs were greater than 6, ranging from 6.08 to 8.81 (Table 1). Based on the predicted results, all ZmCPPs were localized in the nucleus (Table 1), consistent with the characteristics of a transcription factor, and may play a regulatory role in the cell.

Fig. 1.

Fig. 1

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Distribution of ZmCPP genes on maize chromosomes

Table 1.

Characteristics of ZmCPP genes

Mazie GDB ID Gene Name ORF
(bp)		 Protein
Size (aa)		 MW
(kDa)		 pI Subcellular
Location
Zm00001eb031300_T001 ZmCPP1 1185 394 42.99 7.76 Nucleus
Zm00001eb031310_T001 ZmCPP2 2313 770 83.05 6.58 Nucleus
Zm00001eb050860_T002 ZmCPP3 2205 734 78.95 6.18 Nucleus
Zm00001eb135740_T002 ZmCPP4 1185 394 42.46 6.61 Nucleus
Zm00001eb135750_T001 ZmCPP5 2319 772 83.07 7.19 Nucleus
Zm00001eb152420_T001 ZmCPP6 1824 607 66.59 8.45 Nucleus
Zm00001eb237340_T001 ZmCPP7 1593 530 57.88 6.08 Nucleus
Zm00001eb281570_T003 ZmCPP8 1500 499 55.12 7.45 Nucleus
Zm00001eb292380_T001 ZmCPP9 1080 359 39.94 8.29 Nucleus
Zm00001eb301170_T002 ZmCPP10 2403 800 84.98 6.09 Nucleus
Zm00001eb352860_T002 ZmCPP11 1071 356 39.67 8.41 Nucleus
Zm00001eb370470_T002 ZmCPP12 1845 614 67.19 8.81 Nucleus
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Phylogenetic analysis of ZmCPPs

To better understand the evolutionary relationship among ZmCPPs, a Maximum Likelihood (ML) phylogenetic tree was created based on the full-length protein sequences of 12 ZmCPPs, 8 AtCPPs [8], 8 SbCPPs (Table S1), 11 OsCPPs [8], 37 TaCPPs [10], and 9 SiCPPs (Table S1). The phylogenetic tree indicated that the 85 CPPs were divided into four groups (Group I, II, III, and IV) (Fig. 2). Except Group IV, the other three groups both contained 4 ZmCPPs, indicating ZmCPPs could be divided into three subgroups (Fig. 2). Except AtCPPs, the other CPPs in group I, II, III were all from five species (maize, rice, wheat, sorghum, and foxtail millet), revealing that these CPPs may experience similar evolutionary process (Fig. 2). ZmCPPs were more closely related to the CPPs of monocotyledonous plants, such as sorghum and foxtail millet than to Arabidopsis (Fig. 2). In each cluster, the phylogenetic branches of most ZmCPPs exhibited have high bootstrap values with homologous genes in sorghum (Fig. 2), indicating a high sequence homology and a strong ancestral identity of these two species.

Fig. 2.

Fig. 2

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Phylogenetic analysis of CPP homologs in maize (Zm), sorghum (Sb), rice (Os), wheat (Ta), foxtail millet (Si) and Arabidopsis (At). The rootless ML phylogenetic tree building by MEGA 7.0 (10,000 bootstrap replicates) was divided into four groups. Each branch is represented by specific colors

Structural analysis of ZmCPP genes

There were ten conserved motifs (motifs 1–10) in the 12 ZmCPPs (Fig. 3A). The TCR motif was the most conserved one, being present in all ZmCPP proteins (Fig. 3B). Except TCR domain, ZmCPPs in the same branch exhibited similar conserved domains, and there were significant differences in the number and composition of the conserved domains across different branches, which might be attributed to the varying functions of genes across different branches.

Fig. 3.

Fig. 3

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The gene structures and conserved protein motifs of ZmCPPs. The clustering of ZmCPPs is based on the phylogenetic tree shown in Fig. 2. The distribution of the ten conserved motifs (boxes of different colors) in ZmCPPs. (A) The common sequences of motifs 1–10. (B) The location of the TCR motif. (C) The exon-intron structures of ZmCPPs

To evaluate the diversity among ZmCPPs, the gene structure of each ZmCPP was also analyzed. Each ZmCPP contained many exons and introns, and there were significant differences in the introns and exons of ZmCPPs (Fig. 3C). Like the protein domain, the gene structures within the same subfamily were similar (Fig. 3C). Combined, these findings indicated that ZmCPPs from the same branch might exhibit similar functions.

Collinearity among ZmCPPs

The 12 ZmCPPs are unequally distributed on 6 maize chromosomes (Figs. 1 and 4). The occurrence of gene replication events is an important inducer resulting in the expansion of gene family members. We found that the 12 ZmCPPs formed 3 pairs (ZmCPP1 and 4, ZmCPP2 and 5, ZmCPP6 and 12) as a result of gene fragment duplication events (Fig. 4). The remaining ZmCPPs were not identified as fragment or tandem replication pairs (Fig. 4). The relatively low frequency of gene segment duplication events may be the reason for the smaller number of ZmCPP members. No tandem events might also explain the distant distribution of ZmCPPs on maize chromosomes.

Fig. 4.

Fig. 4

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Genome duplication events of ZmCPPs. Schematic diagram of the relationship of ZmCPPs on chromosomes. The heat map in the outer circle represents the gene density of chromosomes. The collinear ZmCPPs are connected by colored lines

Additionally, the synteny among the CPP genes of maize, sorghum, rice, Hordeum vulgare and Arabidopsis was also examined. 12 pairs of orthologous CPP genes in maize and sorghum, 11 pairs in maize and rice, 7 pairs in maize and Hordeum vulgare, and 2 CPP pairs were detected in maize and Arabidopsis (Fig. 5). This finding indicated that CPP genes of maize and sorghum exhibited a relatively close evolutionary relationship.

Fig. 5.

Fig. 5

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The collinearity of ZmCPPs with the CPP genes of sorghum, rice, Hordeum vulgare, and Arabidopsis. The chromosome number is indicated below each chromosome. The gray lines in the background represent the collinear gene pairs between two species, and the CPP collinear genes are connected by blue lines

Cis-acting elements of ZmCPPs

The cis-elements located in promoter regions of ZmCPPs were identified using TBtools. We identified 25 homeotic-acting elements in the promoter regions of ZmCPPs (Fig. 6). Among them, 2 elements (G-box and MRE) were related to light (Fig. 6). Five elements (CAT-box, circadian, MBSI, GCN4-motif, and O2-site) were related to plant growth and development. Five elements (ARE, GC-motif, LTR, MBS, and TC-rich repeats) were involved in plant stress response (Fig. 6). Eight elements (ABA-responsive element (ABRE), AuxRR-core, CGTCA-motif, GARE-motif, P-box, TCA-element, TGACG-motif, and CCAAT-box) were related to the signal pathways of plant and hormone (gibberellic acid, GA and methyl jasmonate, MeJ) interaction. (Fig. 6). These results indicated that ZmCPPs may play significant roles in both plant development as well as in response to environmental stress.

Fig. 6.

Fig. 6

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Analysis of cis-elements in the promoter regions of ZmCPPs. Approximately 2 kb of the upstream promoter region of the ZmCPPs was downloaded from the maize B73 V5 genome

RNA-seq profiles of ZmCPPs

To understand the expression profiles of ZmCPPs, we investigated the tissue-specific expression levels of ZmCPPs in different maize tissues (anthers, root, endosperm, pollen, leaf, internode, meristematic and seed) based on reported RNA-seq data [18, 19]. As shown in Fig. 7, ZmCPP1, ZmCPP2 and ZmCPP5 exhibited higher expression levels in almost all explored tissues, suggesting their special role in maize development. Compared to other tissues, ZmCPPs exhibited lower expression levels in mature pollen, indicating that ZmCPP might be not regulate pollen development (Fig. 7). Furthermore, ZmCPP9, ZmCPP10, and ZmCPP11 were highly expressed in the internode (Fig. 7); Compare to mature leaf, ZmCPP10 showed higher expression level in growing leaf, suggesting it may regulate leaf development (Fig. 7). In addition, like ZmCPP10, ZmCPP1/2/5 also exhibited higher transcript levels in root meristem (Fig. 7), revealed these three genes may modulate root early development. ZmCPP6/7/8/12 from group III (Fig. 1) exhibited similar expression patterns, both showed low or no expression levels in selected tissues (Fig. 7), indicating that these genes were potentially not functional during maize development. Overall, these results indicated that ZmCPPs exhibited functional differentiation during maize growth and development, and might display regulating role in maize vegetative growth and reproductive period.

Fig. 7.

Fig. 7

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Heat map of the expression levels of ZmCPPs in different maize tissues. Tissue name is labeled in figure. Heatmap construction was based on the fragments per kilobase per million mapped reads (FPKM) values. FPKMs data is listed in figure

To explore the expressions of ZmCPPs in response to stress, we downloaded published RNA-seq data of maize B73 in response to abiotic (heat [20], cold [20], salt [20], waterlogging [21], cadmium [22], and UV [20]), biotic (Sugarcane mosaic rirus, SCMV [23]), and nutrition (nitrate) stress [24]. As shown in Fig. 8 and Fig. S1, almost all expression levels of ZmCPPs were downregulated under cold, heat, salt, and root cadmium stresses, suggesting that ZmCPPs might negatively modulate maize response to these stresses. On the contrary, the 12 ZmCPPs were induced by SCMV and waterlogging treatment, indicating ZmCPPs may positively modulate maize response to waterlogging stress and SCMV infection (Fig. 8 and Fig. S1). In addition, ZmCPPs showed different expression pattern under UV stress, and excessive nitrate treatment seemed not obviously change the expression level of ZmCPPs (Fig. S1). Altogether, RNA-Seq analysis indicated a significant role of ZmCPPs in the responses to various environmental stresses, with each ZmCPP having a specific response mechanism.

Fig. 8.

Fig. 8

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Heat map of the expression levels of ZmCPPs in different environmental stresses. Specific stress names are marked in the figure. Heatmap construction was based on the fragments per kilobase per million mapped reads (FPKM) values. Detailed FPKMs are listed in figure

RT-qPCR validation of the expression levels of ZmCPPs under abiotic stress

Based on RNA-Seq analysis, six differentially expressed ZmCPP genes (ZmCPP2/4/8/9/11/12) were further analyzed using RT-qPCR under short-term heat, cold, dehydration, and waterlogging stress. As shown in Fig. 9A and B, the transcript of ZmCPP2/4/9 was induced by heat and drought, while ZmCPP12 was reduced under heat. Like heat shock, ZmCPP2/9 upregulated by cold, and ZmCPP8 showed conversely expression pattern (Fig. 9C). Under waterlogging stress, the expression patterns of both six genes were similar between the RT-qPCR and RNA-Seq data (Figs. 8 and 9D); All genes were upregulated by waterlogging treatment, indicating ZmCPP genes might positively modulating maize waterlogging stress response. Overall, these results indicated that ZmCPPs might be involved in the responses to abiotic stresses (heat, cold, drought, and waterlogging) in maize. However, the precise roles of these genes under abiotic stress need further exploration.

Fig. 9.

Fig. 9

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RT-qPCR analysis of the expressions of ZmCPP2/4/8/9/11/12 in maize B73 seedlings under heat (A), dehydration (B), cold (C), and waterlogging (D) treatments. The treatment times and conditions are labeled within the figure. Values represent means ± SD; n = 3. ** p < 0.01, * p < 0.05 compared with 0 h (Student’s t test)

Subcellular localization and transactivation activity assays of ZmCPP2 and ZmCPP9

ZmCPP2 and ZmCPP9, which positive induced by all four abiotic stresses under RT-qPCR analysis (Fig. 9), were chosen to further explore subcellular localization and transactivation activity. The ZmCPP2/9-GFP or GFP expression vector was transformed into maize mesophyll protoplasts through the PEG-Ca method. Like the GFP control, the ZmCPP2/9-GFP signal was distributed in the cytoplasm and nucleus, indicating that ZmCPP2/9 are widely distributed in cells under normal conditions (Fig. 10A). The transactivation activities of ZmCPP2 and ZmCPP9 were analysis through yeast Y2H system. Like the negative control, yeast cells carrying BD-CPP2/9 and pGADT7 empty vectors did not survive on SD medium without tryptophan, leucine, histidine, and adenine (SD/-LWHA) (Fig. 10B). In contrast, the positive control group could grow well on SD/- LWHA, and could show a blue color after adding X-α-gal (Fig. 10B). This result revealed that ZmCPP2 and ZmCPP9 both had no transcriptional activity in yeast.

Fig. 10.

Fig. 10

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Subcellular localization and transactivation activity assays of ZmCPP2 and ZmCPP9. (A) Subcellular localization of ZmCPP2 and ZmCPP9. The GFP (control) or ZmCPP2/9-GFP fusion expression vectors was transformed into maize mesophyll protoplasts using PEG-Ca method. The GFP signals are labeled green. Bars = 10 μm. (B) Transcription activation analysis of ZmCPP2 and ZmCPP9 in yeast. Full-length ZmCPP2 or ZmCPP9 was fused with the GAL4-DNA BD in the pGBKT7 vector (BD-CPP2 or BD-CPP9). BD-CPP2/9 and pGADT7(AD) were cotransformed into yeast Y2H gold. The yeast cells were cultured on SD lacking tryptophan and leucine (SD-LW), then transferred to an SD medium without tryptophan, leucine, histidine, and adenine (SD-LWHA, containing or not containing X-α-gal) to detect interactions. The positive or negative controls are labeled in the figure, respectively

Discussion

CPP proteins play an important role in plant growth, development and stress response [12]. However, the roles of ZmCPPs in maize have not been reported in detail. Previous report revealed maize contain 13 ZmCPP genes based on B73 V3 genome sequence data, named ZmCPP1-13 [17]. The present study used bioinformatics to identify 12 ZmCPPs in maize last assembled V5 genome (Fig. 1; Table 1). After comparing with our characterization results, we found ZmCPP11 and ZmCPP12 in previous work were located in the same gene locus in our study, and renamed with ZmCPP12 in our work. Due to the differences in the chromosomal position annotation information, ZmCPP1/2/3/13 in the previous studies [17] were named ZmCPP2/3/1/12 in this study, respectively.

The phylogenetic analysis using 85 CPP protein sequences from maize, Arabidopsis, wheat, rice, sorghum, and foxtail millet in the current study showed that these CPPs group into four branches (groupI, II, III, IV) (Fig. 2); Except group IV, ZmCPPs could be divided into other three subfamily with same members in each group (Fig. 2). ZmCPPs have high bootstrap values with SbCPPs (Fig. 2), indicating a strong ancestral identity of these two species; Furthermore, group IV containing AtCPP/OsAPP/TaCPP/SiCPP and lacking ZmCPP/SbCPP; suggesting maize/sorghum, and these other species may have diverged during the evolutionary process, which could have led to CPP protein functional differences. In each subfamily, the number of ZmCPPs is four (Fig. 2). Moreover, the gene and protein structures are highly similar within the same group (Fig. 3), implying that homologous proteins might display similar functions. Previous studies have indicated that AtCPP4 (SOL1)/AtCPP5 (TSO1) (belong to group I in our study) play the same role in controlling floral tissue development [7, 12, 25]; ZmCPP5 showed relative high expression in anthers (Fig. 7) and homologous with AtCPP1/5 (Fig. 2), suggesting ZmCPP5 might also modulating maize inflorescence differentiation.

Gene duplication events drive the expansion of gene families within species; During the evolutionary process, gene duplication enables the genes to display new functions or differentiate from existing functions, thus enhancing plant’s adaptability [26]. Tandem and segmental replications have played important roles in expanding gene families. The present study, through the analysis of gene replication events in the ZmCPP family, revealed that fragment replication plays a crucial role in the evolutionary expansion of ZmCPPs (Fig. 4). The relatively low frequency of duplication events in the ZmCPPs family may be the primary reason why maize has four chromosomes without ZmCPP gene loci and a smaller number of ZmCPP family. CPP gene family were first identified in Arabidopsis [7]. Subsequently, an increasing number of CPPs have been identified and functionally analyzed in multiple species, including rice and soybean [8, 9]. In Arabidopsis, eight CPPs have been identified [8]. In rice, 11 family members have been found [8]. In soybean, the number of CPPs has expanded to 20 [9]. In this study, 12 ZmCPPs were identified (Fig. 1; Table 1), indicating that CPP genes have evolved in different species. Gene duplication is crucial in plant evolution and is also important in driving genome evolution [27, 28]. Homologous genes usually exhibit different functions, but they may also retain the same functions [29, 30]. We identified 12/11/7/2 pairs of orthologous CPPs in maize and sorghum, rice, Hordeum vulgare, and Arabidopsis, respectively (Fig. 5). These results indicate that these gene families have expanded in a species-specific manner. This phenomenon has been widely confirmed in other plant gene families [31–33].

The promoters of ZmCPPs included many cis-elements involve in both plant development as well as in response to environmental stress (Fig. 6). Report indicate some members of plant CPPs play important regulatory roles in the vegetative growth and development of plants. For example, the transcript of CPPs can be specifically expressed/detected in the juvenile leaf stage [9], pollen development stage [7], young leaves, and shoot apices. In Arabidopsis, the mutation of TSO1 could result in changes the structure of flowers and a reduction in pollen levels, and further affects the growth of ovules and pollen [14]. GmCPP16 is specifically expressed in young leaves and root nodules [9]. Transcriptomics analysis showed that ZmCPPs are expressed in various tissues, including roots, seeds, flowers, leaves, and internodes (Fig. 7). Most of these genes exhibit tissue-specific expression; For example, ZmCPP5 has relatively high expression levels in stem internodes and endosperm, while ZmCPP1 is highly expressed in mature leaves. These results revealed that ZmCPPs may be involved in the process of regulating maize growth and development. The expression of plant CPPs are also regulated by various abiotic stresses [34]. Basing on the maize heat treatment (50 ℃ for 4 h) RNA-Seq data [20], we found almost all ZmCPPs were downregulated (Fig. 8); while under 42 ℃, the expression of ZmCPP2/4/9 was induced (Fig. 9). Similar to heat stress, under the treatment condition of 5 °C for 16 h, the expression of almost all ZmCPPs was also downregulated [20]; However, short-term treatment at 4 °C significantly induced the expression of ZmCPP2/9 (Fig. 9). Under heat shock, two AtCPPs (TCX6 and TCX3) are significantly downregulated in atcngc16 knockout mutant [35]. Other published report indicated the transcript of CPPs are modulated by cold stress [36–38]. These results indicated CPPs responds to temperature stress, but the specific response mechanisms still need further research. Recent study reveal ZmCPP2 (in our work named ZmCPP1) positively regulating maize drought tolerance [16]. RT-qPCR result indicated ZmCPP2/4/9 could induced by drought (Fig. 9), moreover ZmCPP1 and ZmCPP4 showed fragment duplication relationship (Fig. 4), implying ZmCPP4 may also involve in maize drought response. All ZmCPPs respond to root waterlogging stress (Figs. 8 and 9), indicating that ZmCPPs might play important role in the oxygen deficiency signaling transduction process in roots compared to other abiotic stresses.

ZmCPP2 and ZmCPP9 which positively induced by short-term heat, cold, drought, and waterlogging stress (Fig. 9), were further chosen to explored the localization and transactivation activity. ZmCPP2/9 both present in the nucleus and cytoplasm under normal conditions (Fig. 10A) and these two proteins seemed unable to activate downstream genes in yeast (Fig. 10B). Although ZmCPP2/9 is induced by stress signals, ZmCPP2/9 may negatively modulating maize stress responses. However, its needs to be further analysis.

Conclusions

In this study, 12 ZmCPPs were identified and characterized. Phylogenetic analysis indicated that ZmCPPs belonged to three subfamilies. Furthermore, ZmCPPs in the same evolutionary group shared their gene and protein structures. The analysis of cis-acting elements in the promoter combined with the expression heatmap based on RNA-seq data indicates ZmCPPs are involved in the regulation of plant growth/development and respond to various biotic/abiotic stresses. RT-qPCR assay further showed ZmCPPs could respond to heat, cold, drought, and waterlogging treatments. Meanwhile, ZmCPP2/9 might play negative regulatory roles in maize stress response. This study provided a theoretical foundation for further exploring the role of ZmCPPs in maize growth regulation and stress response processes.

Methods

Plant material and growth conditions

The Maize (Zea mays L.) inbred line B73 was used in this work. Maize plants were growing in the Crop Resource Repository of Guizhou Normal University (26°22′51.06″N, 106°38′11.80″E, 1100.5 m above sea level) to collect seeds. B73 seeds were grown under greenhouse conditions in the soil at 26 °C/22°C (day/night) and in Hoagland nutrient solution, with a photoperiod of 16 h/8 h (day/night) and continuous cultivation for 2 weeks. During the V3 stage, the seedlings were placed in a 42 ℃ or 4 ℃ growth chamber for either heat or cold stress for 0, 2, 4, or 8 h [39]. The roots were collected for waterlogging by maintaining a 2 cm water layer above the first leaf for 0, 2, 4, or 8 h [39]. For dehydration stress, the seedlings were removed from the soil for 0, 2, 4, or 8 h and harvested the same position of leaves. Three biological replicates were used for downstream analysis at each time point.

Identification of ZmCPP proteins

To identify the ZmCPPs, the maize B73 reference genome (Zm-B73-REFERENCE-NAM-5.0), the sequences of AtCPPs and OsCPPs were downloaded from Maize GDB (https://maizegdb.org, accessed on 18 February 2025), TAIR (https://www.arabidopsis.org/servlets/Search, visited on 18 February 2025) and rice genome annotation venture release 7 (MSU 7.0, http://rice.plantbiology.msu.edu, visited on 18 February 2025), respectively. The hidden Markov model (HMM) files of the core CXC motifs (pfam03638) were acquired from the Pfam database [40] (http://pfam-legacy.xfam.org/, visited on 19 February 2025). The AtCPP/OsCPP sequences as queries to search for ZmCPPs using TBtools (v2.069) (E value: 1e-5) [41]. The sequences containing CXC domain were screened using HMMER 3.0 software [42]. The ZmCPPs identified by the above two methods were combined and removed the repeat sequence, and the complete CXC domain was verified using the Pfam database (http://pfam-legacy.xfam.org/, visited on 20 February 2025) and SMART database [43] (http://smart.embl-heidelberg.de, visited on 20 February 2025). Finally, the members of ZmCPP gene family were obtained and named. Chromosomal localization of the ZmCPPs was obtained from Maize GDB and visualized using TBtools (v2.069).

Phylogenetic analysis and classification of the ZmCPP gene family

Multiple sequence comparisons of full-length plant CPP protein sequences (Table S1) (sorghum, rice, wheat, foxtail millet, Arabidopsis, and maize) (these species represent different plant groups, encompassing the diversity from monocots to dicots) were performed using ClustalW (v1.81) with the default parameters, and the phylogenetic tree was constructed using the MEGA11 software (v11.0.13) [44], in which the Maximum Likelihood (ML) was used. Bootstrap was set to 1000, and other options were set to the default values. The phylogenetic tree was visualized using iTOL (https://itol.embl.de/, accessed on 22 February 2025).

Cis-acting elements analysis and physicochemical properties.

The properties of the ZmCPPs were analyzed using the ExPASy tool (https://web.expasy.org/cgi-bin/protparam/, accessed on 22 February 2025). The subcellular localization prediction website Plant mPloc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 22 February 2025) was used to predict the subcellular localization of ZmCPPs. To determine the cis-elements in the promoters of ZmCPPs, the 2000-bp promoter sequences of ZmCPPs were acquired from the Maize GDB and analyzed using the Plant CARE [45] (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 23 February 2025). Furthermore, TBtools was employed to visualize the composition of cis-elements in the promoters.

Conserved motif and domain analysis

The GFF3 annotation file was downloaded from the Maize GDB database and utilized to display the structures of ZmCPPs in TBtools (v2.069). MEME [46] (http://alternate.meme-suite.org/tools/meme, accessed on 3 March 2025) was used to conduct a conserved motif analysis of ZmCPPs, identifying up to ten conserved motifs. MEME-derived motif sequence logos were presented using TBtools (v2.069).

Gene duplication and synteny analysis of ZmCPPs

Gene duplications of the ZmCPPs were identified and examined by TBtools. The interspecific collinearity of ZmCPPs with the other four species (sorghum, rice, Hordeum vulgare, and Arabidopsis) (these plants are relatively close in evolution, and their genome sizes and structures are relatively moderate, which facilitates detailed collinearity analysis) was used to analyze and visualize using MCScanX [47] and Circos within TBtools, respectively.

RNA-Seq data analysis

To examine the specific expression patterns of ZmCPPs in maize, transcriptome data for different tissues of maize and maize under various stresses were obtained from Maize GDB (https://www.maizegdb.org/, accessed on 19 March 2025). Then, the expressions of ZmCPPs were analyzed and used to create a heatmap using TBtools.

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Three biological duplicate samples were collected for RT-qPCR validation analysis. Total RNA was extracted using Trizol reagent (CWBIO, Beijing, China) and reverse transcribed 2 µg of total RNA sample using a first strand synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) to generate the cDNA. The Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) was used to design gene-specific primers. The 0 h leaf or root samples served as the control, and the relative expression of the gene in each period was calculated using the 2−△△CT method [48]. The ZmActin1 (Zm00001eb348450) was used as the internal control. The significance of the data was confirmed by the student’s t-test (p < 0.05 or p < 0.01) using SPSS v19.0 (SPSS Inc., Chicago, IL, USA). All primers are listed in Table S2.

Subcellular localization and transactivation activity assays

The ORF of ZmCPP2/9 was amplified using primers ZmCPP2/9-Y-F/R (Table S2) and ligated into the pROKII vector [49]. GFP (Empty pROKII vector) control or ZmCPP2/9-GFP vectors was transformed into maize B73 mesophyll protoplasts following a published report [50]. After culturing at 25 ºC for 17 h, GFP signal was observed with a scanning confocal microscope (Andor Revolution WD, Belfast, Northern Ireland, UK).

The ORF of ZmCPP2/9 was amplified using primers (ZmCPP2/9-F/R, Table S2) and ligated into pGBKT7, fused with the Gal4-DNA-binding domain (BD-CPP2/9). The pGADT7 and BD-CPP2/9 vectors were cotransformed into yeast strain Y2HGold. pGBKT7-p53 /pGADT7-largeT and pGBKT7-laminC/pGADT7-largeT were also cotransformed into yeast and used as positive and negative controls, respectively. The transformed yeast was successively cultured in SD/-Trp/-Leu and SD-Trp/-Leu/-His/-Ade/medium with or without X-α-Gal for 4 days.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (13.6KB, xlsx)
Supplementary Material 2 (33.5KB, doc)
Supplementary Material 3 (297.4KB, docx)

Acknowledgements

Not applicable.

Abbreviations

CPP

Cysteine-rich polycomb-like protein

TFs

Transcription factors

CXC

Cys-rich domains

ML

Maximum Likelihood

ORF

Open reading frame

HMM

The hidden Markov model

ABRE

ABA-responsive element

GA

Gibberellic acid

MeJ

Methyl jasmonate

RNA-seq

RNA-sequencing

UV

Ultraviolet

RT-qPCR

Quantitative real-time polymerase chain reaction

Author contributions

LG and YL conceived research plans and designed experiments. LG and TK conducted bioinformatics analysis and experiments. TZ, HW, BZ, and XD analyzed the data. LG and TK wrote the draft. LG and YL reviewed and edited this article. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 32460461]. Guizhou Provincial Basic Research Program (Natural Science) [grant number QKHJCZD (2025) 047]. Guizhou Science and Technology Innovation Team Project [grant number QKHRCCXTD (2025) 055]. Guizhou Province Science and Technology Innovation Platform Construction Project [grant number QKHPTZSYS (2025) 025].

Data availability

All data generated or analyzed during this study were included in this published article and the additional files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lei Gu and Tianyu Kang contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (13.6KB, xlsx)
Supplementary Material 2 (33.5KB, doc)
Supplementary Material 3 (297.4KB, docx)

Data Availability Statement

All data generated or analyzed during this study were included in this published article and the additional files.

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