Identification and analysis of differentially expressed trihelix genes in maize (Zea mays) under abiotic stresses

Plant materials, growth conditions and stress treatments

The experiments were conducted in the Science Park of the Dezhou Academy of Agricultural Science (116.3467E, 37.3600N) and Dezhou University. Maize seeds were preserved at Dezhou Academy of Agricultural Science and Mo17 was more heat tolerant than B73 (Bheemanahalli et al., 2022; Jorgensen et al., 1992). Inbred lines of maize (Zea mays L.) were grown in a greenhouse at 28 °C with a photoperiod of 16 h light and 8 h dark. Seedlings of uniform growth were selected during the flowering stage for tissue expression analysis. We selected the mature root, root tip, spike internode, internode under spike, mature leaf, silks, and female spikelet. For the heat stress treatment, maize seedlings in the three-leaf stage were placed in a light incubator for 4 h at 42 °C, while the control was grown under normal conditions. The above-ground parts were collected immediately. For drought stress treatments, three-leaf-old seedlings of the inbred line B73 were immersed in 20% (w/v) polyethylene glycol 6000 (PEG-6000) solution, and were treated for 4 and 24 h, respectively. The roots of the seedlings were collected for analysis (Jiao et al., 2022; Zeng et al., 2019). Six seedlings were treated per sample, and three biological replicates were conducted for each sample. Figures S1 and S2 show the maize seedlings before and after heat and drought treatment, respectively. All samples were snap-frozen in liquid nitrogen and stored at −80 °C.

Identification and phylogenetic analysis of trihelix family members

The trihelix protein sequences for maize were retrieved from PlantTFDB (http://planttfdb.gao-lab.org/). The trihelix family members were verified using the Pfam database (http://pfam.xfam.org/) and the CDD online tool from the NCBI (https://www.ncbi.nlm.nih.gov/cdd/). A phylogenetic tree was constructed using the neighbor-joining method (NJ) on MEGA7.0 software (Kumar et al., 2018) with bootstrap values from 1,000 replicates.

Analysis expression profiles of trihelix family under heat stress

Genevestigator software (https://genevestigator.com/) (Hruz et al., 2008) was used to compare the maize trihelix genes against the maize GeneChip platform to analyze the transcriptional profile under heat stress. Then, maize seedling expression profiles were downloaded from the NCBI under accession number SRP106663 (Waters et al., 2017). The heat treatment process was as follows: maize seedlings (inbred lines B73, Oh43, and Oh43 × B73) were grown for 14 days under long daylight conditions in a 16/8 h light incubator, then seedlings were placed in an incubator at 50 °C for 4 h. The controls were grown under normal conditions. The cutoff criteria used to select the ZmTHs candidate genes was log2 |fold change| > 1.5.

Chromosomal localization of trihelix family and intra-species collinearity analysis

The chromosome location and structural information of the trihelix family members were obtained from Maize GDB (https://qteller.maizegdb.org/). The intra-species covariance analysis was performed and visualized using TBtools software (Chen et al., 2020) to calculate the values of the duplicated genes with a non-synonymous/synonymous substitution ratio (Ka/Ks). The duplicated genes had greater than a 90% similarity.

Protein characterization and subcellular localization analysis

Using the chromosomal localization information, we renamed the trihelix family members that responded to heat stress (ZmTHs). The protein properties were obtained using the ProtParam (https://web.expasy.org/protparam/) tool in Expasy. Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and SOMPA (http://www.prabi.fr/) were used for the subcellular localization analysis prediction of protein secondary structure.

Conserved motifs, gene structure and promoter analysis of trihelix family

Motif analyses were performed using the online tool MEME (http://memesuite.org/). TBtool software was used to analyze the exon-intron structures within the coding sequences (CDS) and the genomic sequences of each predicted ZmTHs. A total of 2,000 bp promoter sequences were obtained upstream of the CDSs using the sequence extraction tool in TBtools. These were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/) for cis-acting elements analysis. The Gene Structure View tool in TBtools software was used for mapping.

RNA isolation and qRT-PCR analyses of ZmTHs genes

Total RNA was extracted using RNAiso Plus (TaKaRa, Shiga, Japan), and cDNA was obtained by reverse transcription reaction using PrimeScript^TM RT reagent (with gDNA Eraser) (TaKaRa, Shiga, Japan). The solution was diluted 30 times and was stored at −20 °C for later use. Beacon Designer 8.1 was used to design ZmTHs specific primers according to their CDSs (Table 1) and the primers were synthesized using RuiBiotech.

The qRT-PCR reaction system (15 μL) was comprised of 7.5 μL 2×TB Green Premix Ex Taq^TM II (TaKaRa, Shiga, Japan), 0.45 μL forward and reverse specific primers, 1.6 μL ddH₂O, and 5 μL cDNA template. The reaction procedure was pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 10 s for a total of 40 cycles. Each sample was repeated three times and the corresponding Ct values of different samples were detected using the CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA). The relative expression of the target genes were calculated by the 2^−ΔΔCt method after standardization of the internal reference gene ZmActin1 (Livak & Schmittgen, 2001). Data analysis was performed using GraphPad Prism and SPSS software.

Identification of trihelix members and analysis of chromosome localization and intra-species collinearity

A total of 44 trihelix family members were identified in the maize genome, mainly on chromosomes Chr01, Chr02, Chr04, Chr05, and Chr10. Chr05 contained the greatest number, with eight trihelix family members. The remaining five chromosomes had a total of 12 trihelix genes; Chr07 had the smallest distribution with only one gene (Fig. 1). Intra-specific covariance analysis showed that there were 23 pairs of duplicated genes and replication events were the fundamental driving force of trihelix gene evolution. The Ka/Ks of duplicated trihelix gene pairs was less than one (Table 2), suggesting that the maize trihelix genes were subject to purifying selection pressure during evolution.

Analysis of heat stress expression profiles of trihelix members

The results of heat stress expression profiles of trihelix members are shown in Fig. 2. The up-regulation or down-regulation patterns of the trihelix genes in the aboveground tissues of the seedlings were similar between the parents (inbred lines B73, Oh43) and F1 (Oh43 × B73); most of them were down-regulated and only four genes were up-regulated. According to the expression log2 value, 11 trihelix transcription factors with a fold change greater than 1.5 were screened. These were named ZmTH01–ZmTH11 based on the position of their respective genes on the chromosomes (Fig. 1). The expression profile showed that the expression of ZmTH02 and ZmTH08 were up-regulated while the remaining genes were down-regulated after heat stress treatment. These results suggest that ZmTHs may be involved in the regulation of heat stress response in plants.

Evolutionary analysis of the trihelix family genes

The phylogenetic tree results showed that the trihelix proteins of maize, Arabidopsis, and sorghum could be clearly classified into five subfamilies, and that the clustering branches of maize and sorghum were highly similar. The trihelix family members of maize were distributed in all five subfamilies, with the largest subfamily being SIP1. A total of 21 maize trihelix proteins were distributed; the GT-2 subfamily gathered 15, the GTγ subfamily distributed 9, and the GT-1 and SH4 subfamilies each contained six (Fig. 3). ZmTHs were also distributed in every subfamily though the number varied widely. GT-2 had the highest number with eight proteins, SIP1 contained three proteins, GT-1 and SH4 both contained two proteins, and GT-γ contained only one protein (Fig. 4A).

Protein characterization and subcellular localization analysis of ZmTHs

ZmTHs genes were unevenly distributed on chromosomes 1, 2, 4, 5 and 10. Of these, chromosome 10 contained the highest number with four genes (Fig. 1). A total of 16 proteins were encoded, 15 of which were different (Table 3). The protein sequences and physicochemical properties of the ZmTHs transcription factors were quite different. The amino acid length varied from 206–725 amino acids, with the molecular weight ranging from 22.63–76.40 kD. The theoretical pI was 5.24–11.2 with eleven acidic proteins and five basic proteins. The instability index (II) of all ZmTHs proteins was greater than 40, indicating that they were unstable proteins. The grand average of hydropathicity was less than 0, suggesting that they belonged to hydrophilic proteins. Plant-mPLoc localization analysis showed that all ZmTHs proteins were localized in the nucleus. ZmTH04.1 was also distributed in chloroplasts (Table 3).

Secondary structure and conserved motifs analysis of ZmTHs proteins

The secondary structure of the proteins showed that the members of ZmTHs all contained four conformations. The highest proportion of conformations were the random coil and α-helix with an average of 90.51%. Among these two typical conformations, ZmTH02.1 had the highest percentage of 97.11%, and ZmTH09.1 had the lowest percentage, occupying 81.53%. In contrast, the proportion of extended chain and β-angle were relatively low, with an average of 10.49% (Table 4). Conservative motifs analysis showed that most of the closely related members in the phylogenetic tree had the same motifs. These results show that the protein architecture was remarkably conserved within a specific subfamily. MEME software showed that the motifs of ZmTHs of the GT-2 subfamily were the most complex, all members containing motif3, motif6, motif2, motif1, motif9, and the GT-1 subfamily contained motif2, motif1, motif6, motif8 and motif7. The SH4, SIP1 and GT-γ subfamily of the ZmTHs motifs were simpler and motif7 was widely distributed across all proteins (Fig. 4B). The conserved domain of ZmTHs was GT1 or Myb_DNA-bind_4 (Fig. 4C), which was a typical triple helix structure. Five proteins in the GT-2 subfamily contained two triple helix structures, while the remaining had only one (Fig. 4C).

Gene structure and stress-related cis-elements analysis of ZmTHs

The gene structures of ZmTHs were quite different and alternative splicing were observed in Fig. 4E. Among them, ZmTH5.2 of the SIP1, GTγ, and GT-2 subfamilies, contained only one segment of exons with 5′ UTR and 3′ UTR and no introns. The remaining of ZmTHs members all contained introns, and two or more exons, with 5′ UTR or 3′ UTR. The most remarkable feature of ZmTH08.1 was that it contained an extra-long segment of introns of approximately 20,000 bp, which may be related to the regulation of genes expression (Han et al., 2016).

We analyzed the stress-related cis-elements in their promoter regions using the PlantCARE database to better understand the potential regulatory mechanisms of ZmTHs in heat stress. The results showed that there were various stress response cis-elements in ZmTHs promoters, such as high temperature (STRE, TCA), low temperature (LTR), drought (MBS, DRE1), anaerobic (ARE) and wounding (WUN-motif). STRE high temperature stress response elements were predominant, with an average of six per promoter region. In addition, hormone response elements such as salicylic acid, gibberellin, abscisic acid, jasmonic acid, methyl jasmonate, auxin and ethylene were also found in the promoter regions of ZmTHs. There were also tissue-specific expression elements such as meristem, endosperm and root, as well as elements involved in the regulation of maize alcohol protein metabolism (Fig. 4D). These results suggest that the expression of ZmTHs may be regulated by various environmental factors and have different expression specificities in different tissues.

Tissue‑specific expression analysis of ZmTHs at the flowering stage

The expression levels of ZmTHs in the mature root, root tip, spike internode, internode under spike, mature leaf, silks and female spikelet of B73 grown under normal conditions during the flowering stage are shown in Fig. 5. The GT-2 subfamily members, including ZmTH01, ZmTH03, ZmTH05 and ZmTH06, were mainly expressed in roots, especially in the root tip, where the expression was seven to 15 times higher than in ZmActin1. ZmTH02, ZmTH04, ZmTH07, ZmTH09, ZmTH10 and ZmTH11. These genes mostly belong to the GT-1, GTγ and SIP1 subfamilies and had relatively high expressions in the internode. The expression levels of SIP1 subfamily members, including ZmTH02, ZmTH04 and ZmTH10, were higher in female spikelets. The relative expression of the SH4 subfamily members, namely ZmTH08 and ZmTH11, were lower in all tissues except the root tip and were less than 20% of ZmActin1 (Fig. 5). These results were consistent with the fact that the promoter regions of ZmTHs contain root- and meristem-specific expression cis-elements. These results revealed that the expression levels of each subfamily member in different tissues were quite different.

Analysis of expression profiles of ZmTHs under heat and drought stress by qRT-PCR

The expression levels of ZmTHs under heat stress as detected by qRT-PCR are shown in Fig. 6. The results showed that ZmTH08 was up-regulated by heat stress, while ZmTH03, ZmTH04, ZmTH05, ZmTH06, ZmTH07, ZmTH09, ZmTH10 and ZmTH11 were all down-regulated by heat stress in inbred lines B73 and Mo17. These results were consistent with the expression profiles of the ZmTHs in silico. However, ZmTH01 and ZmTH02 were slightly up-regulated in B73 by treatment at 42 °C for 4 h. These results were not identical to the bioinformatics-predicted results of the regulation pattern in leaves treated at 50 °C for 4 h, which were slightly down-regulated by heat stress in Mo17 inbred line. The gene expression patterns of ZmTHs were not completely consistent in various materials treated at different temperatures. These results suggested that ZmTHs respond to heat stress and may have potential functions in abiotic stress.

The analysis of promoter cis-acting elements revealed that ZmTHs contained abundant drought stress response elements, such as MBS and DRE1. qRT-PCR was used to detect the expression of ZmTHs genes under drought stress and the results showed that ZmTH02, ZmTH05, ZmTH06, ZmTH09, and ZmTH11 were up-regulated by drought stress in the inbred line B73. ZmTH06 was especially affected and had expression levels nearly twice those of the untreated genes. The expressions of ZmTH01 and ZmTH07 were down-regulated by drought stress. The expression patterns of the remaining ZmTHs genes subjected to 4 h and 24 h drought treatments were inconsistent. For instance, the expression of ZmTH04 and ZmTH08 were almost unchanged after 4 h drought treatment compared with the control. However, after 24 h of drought treatment, the expression of both trended downward (Fig. 7). These results indicated that ZmTHs may have potential functions in drought stress.