Identification of maize APY genes
Totally, we identified 16 ZmAPY family members from the Ensembl Plant database (https://plants.ensembl.org/index.html), named ZmAPY1-ZmAPY16 (Table S1). Their physicochemical properties, including gene ID, protein size, molecular weight (MW), isoelectric point (pI), the grand averages of hydropathicity (GRAVY), instability index and and localization prediction, were characterized and shown in Table S1. The ZmAPY proteins varied in length from 81 to 701 amino acids, with molecular weights ranging from 8.74822 kDa to 76.9779 kDa. Isoelectric points ranged from 4.70 to 11.62 acidic. Most ZmAPY proteins were hydrophilic (GRAVY < 0) except for ZmAPY6 and ZmAPY7, which were hydrophobic (GRAVY > 0). The estimated instability index ranged from 30.91 to 72.05. Subcellular localization predictions indicated that nine ZmAPY proteins were likely localized in the chloroplast, four in the plasma membrane, three in the mitochondria, one in the extracellular space, and one in the nucleus (Table S1), indicating diverse functional roles for these ZmAPY genes.
To explore the phylogenetic relationships of ZmAPYs with other species, a phylogenetic tree was constructed, incorporating 16 ZmAPYs, 7 AtAPYs, and 9 OsAPYs (Fig. 1A; Table S2). The phylogenetic tree topology classified these APY proteins into three Groups: Group I, II, III. In addition, there are two APY-like proteins (ZmAPYL1, ZmAPYL2) in maize that do not belong to Group I-III. Therefore, we classify them as an outgroup (Fig. 1A, Table S2). The expansion of APYs in maize, compared to AtAPYs and OsAPYs, suggests the potential importance of this gene family to regulate biological processes.
Distribution and collinearity analysis of maize APY genes
To investigate features of the ZmAPYs gene family, we analyzed the chromosome distribution of each ZmAPY gene. Our investigation showed that the ZmAPY genes in the maize genome were unevenly distributed across all 9 chromosomes, with the exception of chromosome 8 (Fig. 1B). The number of APY genes varied on each chromosome. Specifically, there is a single APY gene located on chromosomes Chr5, Chr6, Chr7, and Chr9. Chromosomes Chr2, Chr3, and Chr10 each contain two APY genes, while chromosomes Chr1 and Chr4 each have three APY genes. Gene duplications play a crucial role in the expansion of gene families (Konrad et al. 2011). Segmental duplications lead to the presence of large repetitive chromosomal blocks in the genome and are often associated with chromosomal rearrangements and polyploid events (Lallemand et al., 2020). Colinearity analysis indicated the occurrence of three segmental duplication events involving five ZmAPY genes across the maize genome, while no tandem duplications were observed (Fig. 1B).
APY gene structures and predicted protein motifs
Structural differences in exon-intron arrangement serve as sources of gene family variation and species diversity, leading to alterations in gene expression and function. To investigate the conservation and diversity of gene structure within the maize APY gene family, the exons and introns of 16 APY genes were analyzed based on their coding sequences and genomic data. The number of exons varied among the APY genes, ranging from 1 to 10 (Fig. 2A). Most members contained 6–10 exons, with two members in Group I (ZmAPY5 and ZmAPY6) and one member in Group III (ZmAPY13) having only 1 and 2 exons, respectively (Fig. 2A). The number of introns ranged from 0 to 9. Two transposon insertions of 9.56 kb and 33.57 kb were identified in the intron regions of ZmAPY5 and ZmAPY10, respectively. ZmAPY genes with collinearity exhibited similar gene structures. Additionally, members within the same subgroup typically displayed similar motifs and lengths, suggesting functional similarities (Fig. 2A). Analysis of the protein sequences of all ZmAPY gene family members revealed a conserved GDA1_CD39 domain in all proteins, with varying numbers of transmembrane regions. Specifically, members of Group I had one transmembrane region, most members of Group II had two transmembrane regions, and family members of Group III had no transmembrane regions (Fig. 2B). This structural variation may contribute to the functional distinctions observed among different subgroups.
Expression patterns of APY genes in maize
The investigation into tissue-specific gene expression patterns provides valuable insights into the potential biological roles of the ZmAPY genes. Analysis of the expression patterns within the ZmAPY gene family revealed distinct expression profiles among different members (Fig. 3), highlighting their diverse functions. Specifically, ZmAPY8, ZmAPY9, and ZmAPY12 exhibited specific expression in roots, indicating a potential role for these genes in root-related processes. On the other hand, ZmAPY15 and ZmAPY16 showed significantly higher expression levels in anthers compared to other tissues, suggesting their involvement in anther-related functions. Moreover, there was a notable trend of high expression of ZmAPY11 in seeds and endosperm, implying a potential role in seed development and maturation. Conversely, minimal to no detectable expression was observed for ZmAPY3, ZmAPY4, ZmAPY6, and ZmAPY7 across all tissues and organ. Interestingly, similar tissue-specific expression patterns were observed between collinear ZmAPY genes, such as ZmAPY1/ZmAPY2/ZmAPY5 and ZmAPY15/ZmAPY16, suggesting potential functional conservation or shared regulatory mechanisms among these gene clusters. The differential tissue-specific expression patterns observed among the ZmAPY genes indicate their diverse biological roles and potential contributions to various developmental processes and physiological functions in maize.
Prediction of upstream regulators of APY gene
The analysis of variations in expression patterns among different ZmAPY genes has led to the identification of potential upstream regulators that may control APY gene transcription in maize. By utilizing planttfdb software and existing ChIP-seq data of 104 transcription factors (Tu et al. 2020), a total of 251 upstream regulators were predicted (Fig. 4A, Table. S3). Subsequently, the correlation between the expression levels of these predicted regulators and ZmAPY gene expression was examined. Significantly, NACTF78, ZIM36, bZIP79, and CCHH26 displayed strong correlations with the transcription levels of ZmAPY2 (R2 = 0.51), ZmAPY5 (R2 = 0.56), ZmAPY9 (R2 = 0.67), and ZmAPY14 (R2 = 0.60), respectively (Fig. 4B). Notably, NACTF78 has been previously reported to regulate Fe concentrations in maize kernels, potentially enabling the cultivation of maize varieties with both high yield and high Fe concentrations in their kernels using a molecular marker in the NACTF78 promoter (Yan et al. 2023). Additionally, Vélez-Bermúdez et al. reported that ZML2 (ZIM36) regulates wound-induced lignin genes in maize (Vélez-Bermúdez et al. 2015), while ZmTGA9-1 (bZIP79) has been shown to regulate male sterility in maize (Jiang et al. 2021). The observed correlations between the expression levels of ZmAPY genes and these transcription factors suggest that ZmAPY genes may also play a role in regulating these biological processes, indicating a potential link between APY gene expression and the modulation of Fe concentrations, lignin gene regulation, and male sterility in maize. These findings provide valuable insights into the regulatory network involving ZmAPY genes and their upstream regulators in maize, shedding light on the diverse biological processes influenced by these genes.
Expression analysis of ZmAPYs under drought, cold, heat stresses
To investigate the potential role of APY genes in regulating maize abiotic stress responses, we analyzed the transcription levels of APY genes under drought, cold, and heat stress using RNA-seq data from the maize inbred line B73. Our findings revealed the transcription of the ZmAPY gene is responsive to drought, cold, and heat stress, displaying distinct response profiles (Fig. 5A; 5B). Subsequently, we validated the transcription of ZmAPY genes under drought, cold, and heat stress through qRT-PCR. The results revealed significant upregulation of 6 ZmAPY genes, ZmAPY1, ZmAPY2, ZmAPY8, ZmAPY13, ZmAPY14 and ZmAPY15 under severe drought (DT4), while the transcription of ZmAPY15 was suppressed by drought (Fig. 5C). Cold stress induced the transcription of ZmAPY1 but inhibited the transcription of ZmAPY5, ZmAPY8, ZmAPY11, ZmAPY13 and ZmAPY15 (Fig. 5C). Moreover, heat stress significantly inhibited the transcription of 7 ZmAPY genes, ZmAPY1, ZmAPY2, ZmAPY5, ZmAPY8, ZmAPY11, ZmAPY14, and ZmAPY16, while the transcription of ZmAPY15 was induced by heat (Fig. 5C). It is noteworthy that some of the expression analysis results for the ZmAPY genes are absent from Fig. 5C due to their expression levels falling below the detection threshold of qRT-PCR. The stress-responsive expression patterns of ZmAPY genes suggest their potential regulatory roles in drought, cold, heat, and salt stress responses.
ZmAPYs was associated with agronomic traits and drought resistance of maize
To further investigate the impact of APY on maize agronomic traits and drought resistance, we examined the relationship between SNPs in the ZmAPY gene region and 17 agronomic traits as well as drought phenotypes using the MLM model. Our analysis revealed a significant association between the SNP (chr9_124751424, CC/TT) in ZmAPY16 and maize plant height (Fig. 6A). Subsequent analysis indicated notable differences not only in plant height but also in ear height, drought resistance, and ZmAPY16 expression between the "CC" and "TT" genotypes. Plants with the "TT" allele, showing high ZmAPY6 expression, exhibited greater plant height, ear height, and drought survival rates compared to those with the "CC" allele (Fig. 6B-E), suggesting a positive regulatory role of ZmAPY16 in maize plant height, ear height, and drought resistance. Additionally, we observed a significant association between the SNP (chr1_54070081, CC/TT) in ZmAPY5 and the spinemaking period in maize (Fig. 6F). Plants with the "TT" allele and high ZmAPY5 expression displayed a delayed spinemaking period and enhanced drought survival rates compared to plants with the "CC" allele and low ZmAPY5 expression (Fig. 6G-I), indicating a negative regulation of maize spinemaking and a positive regulation of maize drought resistance by ZmAPY5.
Genetic variation within ZmAPYs regulate the content of drought-induced metabolites
Metabolites, as small molecules that serve as the end products of metabolic processes and physiological pathways, are known to play crucial roles in plant drought resistance (Kim et al. 2017; Todaka et al. 2017). These compounds can act as osmoprotectants, antioxidants, signaling molecules, and regulators of various stress-responsive pathways in plants (Nakabayashi et al. 2014; Obata et al. 2013; Fàbregas et al. 2018). Analyzing the genome-wide metabolite profiles of 385 maize natural inbred lines grown under well-watered and drought-stressed conditions (Zhang et al. 2021a), we identified metabolite quantitative trait loci (mQTL) for 18 metabolites that co-located with the ZmAPY genes, indicating a potential relationship between ZmAPYs and the levels of these metabolites (Fig. 7A). Further investigation into these metabolites revealed that four drought-induced metabolites were influenced by genetic variations within the ZmAPYs gene region. Specifically, genetic variations chr1.S_65348503 and chr1.S_65352471 within the ZmAPY15 gene region were found to regulate the contents of metabolites PN_group_05106 and PN_group_17082, while another genetic variation chr1.S_65352471 within the ZmAPY11 gene region was associated with the regulation of the metabolite PN_group_00505. Additionally, a genetic variation chr10.S_71730551 within the ZmAPY7 gene region was linked to the regulation of the metabolite PN_group_11670 (Fig. 7B). These findings suggest that ZmAPY genes may impact maize drought resistance by modulating the contents of these drought-induced metabolites. This insight highlights the potential role of ZmAPY genes in mediating maize response to drought stress through the regulation of key metabolites involved in stress adaptation and tolerance.