A Comprehensive Identification and Expression Analysis of the WUSCHEL Homeobox-Containing Protein Family Reveals Their Special Role in Development and Abiotic Stress Response in Zea mays L.

Maize is an important food and cash crop worldwide. The WUSCHEL (WUS)-related homeobox (WOX) transcription factor (TF) family plays a significant role in the development process and the response to abiotic stress of plants. However, few studies have been reported on the function of WOX genes in maize. This work, utilizing the latest maize B73 reference genome, results in the identification of 22 putative ZmWOX gene family members. Except for chromosome 5, the 22 ZmWOX genes were homogeneously distributed on the other nine chromosomes and showed three tandem duplication and 10 segmental duplication events. Based on phylogenetic characteristics, ZmWOXs are divided into three clades (e.g., WUS, intermediate, and ancient groups), and the majority of ZmWOXs in same group display similar gene and protein structures. Cross-species collinearity results indicated that some WOX genes might be evolutionarily conservative. The promoter region of ZmWOX family members is enriched in light, plant growth/hormone, and abiotic stress-responsive elements. Tissue-specific expression evaluation showed that ZmWOX genes might play a significant role in the occurrence of maize reproductive organs. Transcriptome data and RT-qPCR analysis further showed that six ZmWOX genes (e.g., ZmWOX1, 4, 6, 13, 16, and 18) were positively or negatively modulated by temperature, salt, and waterlogging stresses. Moreover, two ZmWOXs, ZmWOX1 and ZmWOX18, both were upregulated by abiotic stress. ZmWOX18 was localized in the nucleus and had transactivation activities, while ZmWOX1 was localized in both the cytoplasm and nucleus, without transactivation activity. Overall, this work offers new perspectives on the evolutionary relationships of ZmWOX genes and might provide a resource for further detecting the biological functions of ZmWOXs.


Introduction
The development of apical meristematic tissue and the differentiation of lateral organs affect the plant shape and height.The homeobox (HB) gene family is a large group of plants (containing 14 subfamilies).Mutations in these classes of genes can lead to embryonic or other periods of developmental abnormalities [1].The WUSCHEL-related homeobox (WOX) TF gene group is among the most important HB families.The protein N-terminus of its family members contains a conserved DNA-binding region consisting of three αhelices (e.g., homeodomain and HD, including 60-66 amino acids) [2].In addition to the conservative HD motif, the WOX protein also contains some functional domains in the C-terminal, such as the typical WUS-box structural domain (TLXLFP, seven amino acids, X indicates any amino acid), EAR motif (ERF-associated amphiphilic repression), and acidic domain [3,4].The WOX proteins in many species may be divided into three groups based on their structural similarity: ancient, intermediate, and modern/WUS [5].Ancient WOX genes are widely distributed in plants.Intermediate group members are found in vascular plants.Finally, WUS exists only in seed plants [6].
In addition to playing a crucial regulatory role in plant development, WOX TFs could also be modulated by various abiotic stresses (e.g., drought, low temperature, and salt) [32].In rice, OsWOX11 regulates the development process of root hair to increase rice drought tolerance [31].Eight OsWOX genes, including OsWUS, positively responded to drought stress.For instance, OsWOX3 and OsWOX5 could be induced by salt, while four members of OsWOX respond positively to cold [29].Some WOX gene family members in cotton and cucumber also respond positively or negatively to several environmental stresses [18,33].The OsWOX13 gene is modulated by drought, cold, and salt.Overexpression of OsWOX13 in rice significantly enhanced plant drought stress tolerance [34].Overexpression of MdWOX13-1 also enhanced plant drought stress resistance by activating the reactive oxygen species scavenging system [35].
Maize is an important crop worldwide.Previous studies have shown 21 ZmWOX genes in maize based on ancient genome data [16].Four ZmWOX genes (e.g., ZmNS1, ZmNS2, ZmWUS1, and ZmWUS2) were also involved in leaf development and promoted somatic embryogenesis in maize.Double mutants of ZmNS1 and ZmNS2 result in narrow leaf width but unchanged leaf length [36].Both ZmWUS1 and ZmWUS2 are homologous to AtWUS.At the same time, the expression patterns of these two genes have significant differences; the transcript of ZmWUS1 is in dynamic change during the nutrient growth stage, and the ZmWUS2 gene is specifically expressed in young leaf primordia [37].Meanwhile, co-overexpression of ZmWUS2 and BBM genes in maize notably enhances somatic embryogenesis and shortens the time of genetic transformation [38].Despite the above findings, the function of ZmWOX genes in maize development and the abiotic stress response is largely unknown.
In this work, we identified 22 WOX genes in maize and conducted a comprehensive analysis of these genes according to the newly assembled maize B73 reference genome (Zm-B73-REFERENCE-NAM-5.0).The expression level of ZmWOXs under normal and abiotic conditions was also explored.Meanwhile, the subcellular localization and transcriptional activation activity of ZmWOX1 and ZmWOX18 were analyzed.This study provides new information about the evolutionary relationships of the ZmWOX genes and may help further understand the possible functions of ZmWOXs.

Identification and Characterization of ZmWOX Genes
A 2010 study reported a maize genome containing 21 WOX genes [16].In this study, based on a new version of the maize B73 genome (NAM-5.0), a homology search was performed using known AtWOX and OsWOX sequences [15,16] as detectors.After removing duplicates and confirming the conservative HD domain, 22 putative WOX genes were identified in maize (Table 1).The detailed location site of each ZmWOX gene was obtained after searching the genome (Supplementary Table S1).The 22 ZmWOX genes were labeled ZmWOX1 to ZmWOX22 according to their location in the chromosomal region (Supplementary Table S1).Except for chromosome 5, the other nine chromosomes all contained ZmWOXs.Chromosome 8 had the most ZmWOX genes (five members), followed by four members located on chromosome 3, while chromosomes 1, 7, and 9 only included one ZmWOX gene, respectively (Supplementary Table S1).The coding sequence (CDS) of ZmWOXs ranged from 252 bp (ZmWOX8) to 1551 bp (ZmWOX7), and 22 ZmWOX proteins contained 83-516 amino acids (aa) (average: 287 aa) (Table 1).The isoelectric points (PI) and molecular weights of the ZmWOXs ranged from 5.56 to 10.52 (average: 8.00) and 10.01 to 53.26 kDa (average: 30.67), respectively (Table 1).According to the predicted results of the website, most ZmWOX proteins (17 members) were distributed in the nucleus (Table 1), consistent with the localization of transcription factors (TF).The other five members were distributed in the chloroplast (three members) and mitochondrion (two members), respectively (Table 1).It may be responsible for the transcriptional regulation of genes in plastids.

Structural Analysis of ZmWOX Genes
motifs were only present in special groups, reflecting the diversity of ZmWOXs.The intermediate clade members contained motifs 3, 4, 6, 9, and 10.Meanwhile, both members included motifs 3-4 in the C terminal, except for ZmWOX8 (short sequence) (Figure 2A).ZmWOX6 and ZmWOX18 (ancient members) also contained motif 4 at the N-terminus (Figure 2A).In Arabidopsis, WUS subfamily members contained two conserved domains: WUS box and EAR-like.All members contained the WUS box, while the EAR motif was found only in AtWUS [39].As shown in Figure 2A, except for ZmWOX9, 11, and 14 (short sequence), motif 5 (WUS box, TLXLFP, Figure S1) was identified in all maize WUS members.We also found the EAR domain in ZmWOX2 (ZmWUS1) and ZmWOX22 (ZmWUS2) (Figure S2).
We analyzed the exon-intron distribution of ZmWOX genes using TBtools.Except for ZmWOX9 and ZmWOX11, which had only one exon and no intron, all ZmWOXs contained at least two exons and one intron (Figure 2B).The exon and intron lengths were similar in the same clade.Still, it displayed wide variation between the different subfamilies, mainly due to the differences in the intron sequence (Figure 2B).The intermediate members contained longer exon sequences than the WUS and ancient clades (Figure 2B), leading to longer protein sequences (Figure 2A).Overall, these results revealed that the same clade of ZmWOX genes shows less variation in gene and protein structure and may display the same functions.We analyzed the exon-intron distribution of ZmWOX genes using TBtools.Except for ZmWOX9 and ZmWOX11, which had only one exon and no intron, all ZmWOXs contained at least two exons and one intron (Figure 2B).The exon and intron lengths were similar in the same clade.Still, it displayed wide variation between the different subfamilies, mainly due to the differences in the intron sequence (Figure 2B).The intermediate members contained longer exon sequences than the WUS and ancient clades (Figure 2B), leading to longer protein sequences (Figure 2A).Overall, these results revealed that the same clade of ZmWOX genes shows less variation in gene and protein structure and may display the same functions.

Gene Duplication and Synteny Analysis of the ZmWOX Genes
Except for chromosome 5, the 22 ZmWOXs were widely distributed in the remaining nine chromosomes (Supplementary Table S1 and Figure 3).Some ZmWOX genes were located close together and formed three tandem duplication events (ZmWOX4/5/6, ZmWOX16/17/18, and ZmWOX21/22) (Figure 3).According to the gene duplication events analysis, except for three tandem duplication events (Figure 3), 10 pairs of segmental duplication (homologous) in ZmWOX genes were also identified in the maize genome (Figure 3).Among these 10 pairs, five belonged to the WUS family (e.g., ZmWOX2/22, ZmWOX3/9, ZmWOX4/16, ZmWOX5/17, and ZmWOX10/14), four in the intermediate clade (e.g., ZmWOX1/13, ZmWOX1/19, ZmWOX7/12, and ZmWOX7/15), and only ZmWOX6/18 in the ancient group (Figure 3).These results indicate that segmental duplication may be the main force driving the extension of the ZmWOXs family.A collinear analysis between maize and the other four species (i.e., wheat, sorghum, rice, and Arabidopsis) was constructed to further understand the evolutionary mechanism of the ZmWOX genes.As shown in Figure 4, an amount of 40, 17, 16, and four collinear gene pairs (a total of 77) were identified between maize and wheat, maize and sorghum, maize and rice, and maize and Arabidopsis, respectively.This result indicated that the A collinear analysis between maize and the other four species (i.e., wheat, sorghum, rice, and Arabidopsis) was constructed to further understand the evolutionary mechanism of the ZmWOX genes.As shown in Figure 4, an amount of 40, 17, 16, and four collinear gene pairs (a total of 77) were identified between maize and wheat, maize and sorghum, maize and rice, and maize and Arabidopsis, respectively.This result indicated that the amount of WOX homologous genes is high in crops and that paralogous homologous genes may play a significant role in the WOX family evolution process.ZmWOX4 (Chr 3), 7 (Chr 3), 14 (Chr 8), and 17 (Chr 8) showed homologous pairs in all five plants (Figure 4), indicating that these four genes may have existed before the evolution of these five plant species.The results suggest that the ZmWOX family genes may have similar functions to the WOXs of other species.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 19 amount of WOX homologous genes is high in crops and that paralogous homologous genes may play a significant role in the WOX family evolution process.ZmWOX4 (Chr 3), 7 (Chr 3), 14 (Chr 8), and 17 (Chr 8) showed homologous pairs in all five plants (Figure 4), indicating that these four genes may have existed before the evolution of these five plant species.The results suggest that the ZmWOX family genes may have similar functions to the WOXs of other species.

The Cis-Element Analysis of the ZmWOX Promoter
A 2 kb sequence upstream of the start codon of each gene was downloaded from the maize genome NAM 5.0 and submitted to the online PlantCARE database to detect the diversity of cis-elements present in the promoter regions of the ZmWOX genes.As shown in Figure 5 and Supplementary Table S3, except for the TATA box and CAAT box, 1037 elements were found in the promoter of 22 ZmWOXs.These elements could be divided into four classes: light responsive (e.g., Box 4, G-box, I-box, AE-box, MRE, Sp1, and Gap-box), hormones responsive (e.g., ABA-responsive elements (ABRE), TCA-element, TGA-element, TATC-box, P-box, TCA, ERE, and AuxRR-core), plant growth (e.g., ARE, CAT-box, circadian, MBSI, MSA-like, O2-site, AT-rich element, and RY-element), and stress-responsive elements (e.g., STRE, Myc, MYB site, W box, MBS, LTR, drought-responsive elements (DRE) core, and TC-rich repeats).Some elements were widely located in the ZmWOXs promoter,

The Cis-Element Analysis of the ZmWOX Promoter
A 2 kb sequence upstream of the start codon of each gene was downloaded from the maize genome NAM 5.0 and submitted to the online PlantCARE database to detect the diversity of cis-elements present in the promoter regions of the ZmWOX genes.As shown in Figure 5 and Supplementary Table S3, except for the TATA box and CAAT box, 1037 elements were found in the promoter of 22 ZmWOXs.These elements could be divided into four classes: light responsive (e.g., Box 4, G-box, I-box, AE-box, MRE, Sp1, and Gap-box), hormones responsive (e.g., ABA-responsive elements (ABRE), TCAelement, TGA-element, TATC-box, P-box, TCA, ERE, and AuxRR-core), plant growth (e.g., ARE, CAT-box, circadian, MBSI, MSA-like, O2-site, AT-rich element, and RY-element), and stress-responsive elements (e.g., STRE, Myc, MYB site, W box, MBS, LTR, droughtresponsive elements (DRE) core, and TC-rich repeats).Some elements were widely located in the ZmWOXs promoter, such as ABRE (contained in 20 members), MYB/MYC bind-ing site elements related to abiotic stress and plant growth (contained in all members), and STRE elements (18 ZmWOXs) (Figure 5 and Supplementary Table S3).These results suggest that the ZmWOX genes might be involved in stress resistance and the maize growth/hormone pathway.S3).These results suggest that the ZmWOX genes might be involved in stress resistance and the maize growth/hormone pathway.
Figure 5.The cis-element analysis in promoter regions of ZmWOX genes.A promoter region of about 2 kb upstream of the ZmWOX genes was downloaded from the genome for analysis.Different colors represent different genes.The cis-element names and counts are listed in Supplementary Table S3.S3.

Expression Patterns of ZmWOX Genes in Different Maize Tissues
Published RNA-seq datasets [40] (Supplementary Table S4) from 15 maize tissues with varied developmental periods were used to explore the expression patterns of ZmWOX genes in the maize growth and development stage.As shown in Figure 6, except for ZmWOX8 and ZmWOX11, all tissues had no expression.The other 20 genes showed high expression levels in specific tissues, especially for ZmWOX6 and 18 displayed high expression levels in all tissues.Meanwhile, ZmWOXs in reproductive organs (e.g., ear, embryo, inflorescence, and tassel) showed relatively higher transcript amounts compared to nutrient tissue (e.g., leaf, stem, and root) (Figure 6).Except for ZmWOX6, 16, 18, 21, and 22, the peak expression level of the other genes was in all reproductive tissues (Figure 6).For example, the expression of ZmWOX1 in embryo was almost triple as high as that in the roots (Figure 6).These results indicate that ZmWOXs might play an important role in maize development, especially in the occurrence of reproductive organs.

Expression Patterns of ZmWOX Genes in Different Maize Tissues
Published RNA-seq datasets [41] (Supplementary Table S4) from 15 maize tissues with varied developmental periods were used to explore the expression patterns of ZmWOX genes in the maize growth and development stage.As shown in Figure 6, except for ZmWOX8 and ZmWOX11, all tissues had no expression.The other 20 genes showed high expression levels in specific tissues, especially for ZmWOX6 and 18 displayed high expression levels in all tissues.Meanwhile, ZmWOXs in reproductive organs (e.g., ear, embryo, inflorescence, and tassel) showed relatively higher transcript amounts compared to nutrient tissue (e.g., leaf, stem, and root) (Figure 6).Except for ZmWOX6, 16, 18, 21, and 22, the peak expression level of the other genes was in all reproductive tissues (Figure 6).For example, the expression of ZmWOX1 in embryo was almost triple as high as that in the roots (Figure 6).These results indicate that ZmWOXs might play an important role in maize development, especially in the occurrence of reproductive organs.S4).The name of the tissues is labeled in the Figure .SAM: shoot apical meristem.

Expression Patterns of ZmWOXs under Abiotic Stress Conditions
We used the published transcriptome data about drought [42] (Supplementary Table S5), salt [43] (Supplementary Table S5), temperature [44] (Supplementary Table S5), and waterlogging [40] (Supplementary Table S5) stresses to analyze the expression patterns of ZmWOX genes under abiotic stresses.As shown in Figure 7, ZmWOX6 was downregulated by heat and cold treatments.ZmWOX13 and ZmWOX22 (ZmWUS2) positively responded to cold stress, while cold stress inhibited ZmWOX17.The other members of ZmWOXs did not seem to be influenced by temperature stress (Figure 7).For salt stress, whether in salt-stress-sensitive or salt-tolerant maize lines, ZmWOX1, ZmWOX2, ZmWOX6, ZmWOX13, and ZmWOX18 were positively modulated by salt stress (Figure  S4).The name of the tissues is labeled in the Figure .SAM: shoot apical meristem.

Expression Patterns of ZmWOXs under Abiotic Stress Conditions
We used the published transcriptome data about drought [41] (Supplementary Table S5), salt [42] (Supplementary Table S5), temperature [43] (Supplementary Table S5), and waterlogging [44] (Supplementary Table S5) stresses to analyze the expression patterns of ZmWOX genes under abiotic stresses.As shown in Figure 7, ZmWOX6 was downregulated by heat and cold treatments.ZmWOX13 and ZmWOX22 (ZmWUS2) positively responded to cold stress, while cold stress inhibited ZmWOX17.The other members of ZmWOXs did not seem to be influenced by temperature stress (Figure 7).For salt stress, whether in saltstress-sensitive or salt-tolerant maize lines, ZmWOX1, ZmWOX2, ZmWOX6, ZmWOX13, and ZmWOX18 were positively modulated by salt stress (Figure 7).For waterlogging maize root stress, except for ZmWOX4 and 6 (downregulated by waterlogging), the other 20 ZmWOXs were all induced by the waterlogging stress (Figure 7), implying that these genes might show a key function in maize root response to low oxygen stress.Unlike the above three abiotic stresses, the ZmWOXs did not seem to respond to drought stress (Figure 7), even though some of the promoters of these genes contained DRE (Figure 5 and Supplementary Table S3).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 10 of 19 7).For waterlogging maize root stress, except for ZmWOX4 and 6 (downregulated by waterlogging), the other 20 ZmWOXs were all induced by the waterlogging stress (Figure 7), implying that these genes might show a key function in maize root response to low oxygen stress.Unlike the above three abiotic stresses, the ZmWOXs did not seem to respond to drought stress (Figure 7), even though some of the promoters of these genes contained DRE (Figure 5 and Supplementary Table S3).S5).For salt stress, 'S' indicates a salt-sensitive maize-inbred line (L29).'T' indicates a salt-tolerant maizeinbred line (L87).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

RT-qPCR Analysis of ZmWOX Genes under Abiotic Stresses
ZmWOX1, 4, 6, 13, 16, and 18 were simultaneously positively or negatively modulated by temperature, salt, and waterlogging stresses (Figure 7).These genes were chosen to further explore the expression levels of ZmWOX genes in heat, cold, salt, and waterlogging stresses in treated maize lines using RT-qPCR.ZmWOX1, ZmWOX16, and ZmWOX18 were induced by heat shock, and ZmWOX6 and 13 were downregulated by heat and cold stress.In contrast, ZmWOX4 did not respond to temperature stress (Figure 8).For salt stress (200 mM NaCl), except for ZmWOX4 (no change), the transcript levels of the other five genes were upregulated by salt stress (Figure 8).Although ZmWOX4 was not responsible for temperature and salt stresses, the expression level of this gene was reduced under waterlogging treatment (Figure 8).Low oxygen stress also induced the other four genes (ZmWOX1, 13, 16, and 18) or downregulated ZmWOX6.Overall, six ZmWOX genes displayed different expression patterns in abiotic stresses.S5).For salt stress, 'S' indicates a salt-sensitive maize-inbred line (L29).'T' indicates a salt-tolerant maize-inbred line (L87).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

RT-qPCR Analysis of ZmWOX Genes under Abiotic Stresses
ZmWOX1, 4, 6, 13, 16, and 18 were simultaneously positively or negatively modulated by temperature, salt, and waterlogging stresses (Figure 7).These genes were chosen to further explore the expression levels of ZmWOX genes in heat, cold, salt, and waterlogging stresses in treated maize lines using RT-qPCR.ZmWOX1, ZmWOX16, and ZmWOX18 were induced by heat shock, and ZmWOX6 and 13 were downregulated by heat and cold stress.In contrast, ZmWOX4 did not respond to temperature stress (Figure 8).For salt stress (200 mM NaCl), except for ZmWOX4 (no change), the transcript levels of the other five genes were upregulated by salt stress (Figure 8).Although ZmWOX4 was not responsible for temperature and salt stresses, the expression level of this gene was reduced under waterlogging treatment (Figure 8).Low oxygen stress also induced the other four genes (ZmWOX1, 13, 16, and 18) or downregulated ZmWOX6.Overall, six ZmWOX genes displayed different expression patterns in abiotic stresses.

Subcellular Localization and Transactivation Activity Assays of ZmWOX1 and 18
ZmWOX1 and ZmWOX18, exhibiting positive response patterns under RT-qPCR analysis (Figure 8), were chosen to explore subcellular localization and transactivation activity to further determine the functions of ZmWOX genes.The 35S∷ZmWOX1/18-GFP and GFP empty vector (control) were cotransformed with the nucleus marker vector (mCherry) into tobacco leaves using the Agrobacterium-mediated method.The fluorescence signals of GFP and ZmWOX1-GFP were distributed in the cell membrane, cytoplasm, and nucleus.In contrast, the fluorescence signal of the ZmWOX18-GFP was only distributed in the nucleus, indicating that ZmWOX18 is localized in the nucleus, and ZmWOX1 was widely distributed in cells (Figure 9A).The transactivation activities of ZmWOX1 and ZmWOX18 were assessed using the Y2H yeast system.Like the positive control, yeast cells carrying pGBKT7-ZmWOX18 (BD-WOX18) and pGADT7 empty vectors grew well on an SD medium without tryptophan, leucine, histidine, and adenine (SD/-Trp/-Leu/-Ade/-His).They could show a blue color after adding X-α-gal.In contrast, the negative control and BD-WOX1+AD groups did not survive (Figure 9B).This result revealed that ZmWOX18 had transcriptional activity in yeast, while ZmWOX1 showed no transcriptional activity.

Subcellular Localization and Transactivation Activity Assays of ZmWOX1 and 18
ZmWOX1 and ZmWOX18, exhibiting positive response patterns under RT-qPCR analysis (Figure 8), were chosen to explore subcellular localization and transactivation activity to further determine the functions of ZmWOX genes.The 35S∷ZmWOX1/18-GFP and GFP empty vector (control) were cotransformed with the nucleus marker vector (mCherry) into tobacco leaves using the Agrobacterium-mediated method.The fluorescence signals of GFP and ZmWOX1-GFP were distributed in the cell membrane, cytoplasm, and nucleus.In contrast, the fluorescence signal of the ZmWOX18-GFP was only distributed in the nucleus, indicating that ZmWOX18 is localized in the nucleus, and ZmWOX1 was widely distributed in cells (Figure 9A).The transactivation activities of ZmWOX1 and ZmWOX18 were assessed using the Y2H yeast system.Like the positive control, yeast cells carrying pGBKT7-ZmWOX18 (BD-WOX18) and pGADT7 empty vectors grew well on an SD medium without tryptophan, leucine, histidine, and adenine (SD/-Trp/-Leu/-Ade/-His).They could show a blue color after adding X-α-gal.In contrast, the negative control and BD-WOX1+AD groups did not survive (Figure 9B).This result revealed that ZmWOX18 had transcriptional activity in yeast, while ZmWOX1 showed no transcriptional activity.
ZmWOX1/18-GFP and GFP empty vector (control) were cotransformed with the nucleus marker vector (mCherry) into tobacco leaves using the Agrobacterium-mediated method.The fluorescence signals of GFP and ZmWOX1-GFP were distributed in the cell membrane, cytoplasm, and nucleus.In contrast, the fluorescence signal of the ZmWOX18-GFP was only distributed in the nucleus, indicating that ZmWOX18 is localized in the nucleus, and ZmWOX1 was widely distributed in cells (Figure 9A).The transactivation activities of ZmWOX1 and ZmWOX18 were assessed using the Y2H yeast system.Like the positive control, yeast cells carrying pGBKT7-ZmWOX18 (BD-WOX18) and pGADT7 empty vectors grew well on an SD medium without tryptophan, leucine, histidine, and adenine (SD/-Trp/-Leu/-Ade/-His).They could show a blue color after adding X-α-gal.In contrast, the negative control and BD-WOX1+AD groups did not survive (Figure 9B).This result revealed that ZmWOX18 had transcriptional activity in yeast, while ZmWOX1 showed no transcriptional activity.

Subcellular Localization and Transactivation Activity Assays of ZmWOX1 and 18
ZmWOX1 and ZmWOX18, exhibiting positive response patterns under RT-qPCR analysis (Figure 8), were chosen to explore subcellular localization and transactivation activity to further determine the functions of ZmWOX genes.The 35S∷ZmWOX1/18-GFP and GFP empty vector (control) were cotransformed with the nucleus marker vector (mCherry) into tobacco leaves using the Agrobacterium-mediated method.The fluorescence signals of GFP and ZmWOX1-GFP were distributed in the cell membrane, cytoplasm, and nucleus.In contrast, the fluorescence signal of the ZmWOX18-GFP was only distributed in the nucleus, indicating that ZmWOX18 is localized in the nucleus, and ZmWOX1 was widely distributed in cells (Figure 9A).The transactivation activities of ZmWOX1 and ZmWOX18 were assessed using the Y2H yeast system.Like the positive control, yeast cells carrying pGBKT7-ZmWOX18 (BD-WOX18) and pGADT7 empty vectors grew well on an SD medium without tryptophan, leucine, histidine, and adenine (SD/-Trp/-Leu/-Ade/-His).They could show a blue color after adding X-α-gal.In contrast, the negative control and BD-WOX1+AD groups did not survive (Figure 9B).This result revealed that ZmWOX18 had transcriptional activity in yeast, while ZmWOX1 showed no transcriptional activity.
ZmWOX1/18-GFP fusion expression vectors were cotransformed with mCherry (nuclear marker) into tobacco leaves using an Agrobacterium-mediated method.The GFP, mCherry, and chlorophyll autofluorescence (ChI) signals are labeled green, red, and purple, respectively.The empty GFP vector was used as a control.GFP: green fluorescent protein.Bars = 50 µm.(B) Transcription activation analysis of ZmWOX1 and ZmWOX18 in yeast.Full-length ZmWOX1 or ZmWOX18 was fused with the GAL4-DNA BD in the pGBKT7 vector (BD-WOX1 or BD-WOX18).BD-WOX1/18 and pGADT7(AD) were cotransformed into yeast Y2H gold.The yeast cells were cultured on a synthetic dextrose medium (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.
The 22 ZmWOX genes were widely distributed on the genome except chromosome 5 (Supplementary Table S1 and Figure 3).Gene duplication (containing tandem and segmental events) is a broad phenomenon in the evolution of plant gene family members [46].In this work, three tandem and 10 segment events were identified in ZmWOX genes (Figure 3), indicating that the relatively high-segmental duplications in the maize WOX gene family might be the main driving forces for the expansion of ZmWOXs.In other species, such as sunflower [14], melon [10], cotton [13], and Sacred Lotus [11], the main driving forces for increasing the WOX members are also segmental duplications, indicating the WOX family in plants might prefer using segmental duplications in the evolutionary process.Notably, ZmWOX8, 11, 20, and 21 were not included in gene duplication events (Figure 3), although ZmWOX20/21 showed high expression levels in embryo, stem, and submergence stress (Figures 6 and 7).ZmWOX8 and 11 have almost no expression in maize development and abiotic stress response (Figures 6 and 7), suggesting that these two genes might be the ancient and silenced genes.Evolutionary relationship analysis using WOX protein full-length sequences of maize and four other plant species (e.g., sorghum, wheat, rice, and Arabidopsis) indicated that the maize WOX family was divided into three clades (e.g., WUS, intermediate, and ancient) (Figure 1).This condition was consistent with the classification of the WOX family in other species [5].In addition to the evolutionary tree, we also used cross-species collinearity analysis to explore the relationship of the WOX family.A high homology was observed between maize and the other three Poaceae plants (Figure 4).Although there was a low collinear relationship between maize and Arabidopsis, there were also two pairs of homologous genes in these two species (Figure 4).These results indicate that WOX genes may undergo conservative evolutionary progress in plants and might have similar biological functions among different species.Notably, ZmWOX8 and 9 on chromosome 4, without relationships with the other four plants (Figure 4), imply that some WOXs might have special features only displayed in their species.
All ZmWOXs containing the typical HD conserve domain (motifs 1 and 2) (Figure 2) and members in the same groups display similar gene and protein structures (Figures 1 and 2).This reveals that different group members might show different functionalities.In other plants, WOX members in the same evolutionary group also display similar gene and protein structures, though with greater structural differences in different groups [10,11,13,14], suggesting that structural differences may account for functional differences of WOX genes in the same or different species.
In plants, the WOX genes mainly participate in plant growth and development, for example, in the maintenance and stabilization of SAM [23], RAM [47], inflorescence [48], and vascular [27] tissues.We found many cis-acting elements related to light, development, hormones, and abiotic stress response located in the promoter region of ZmWOX genes (Figure 5 and Supplementary Table S3).Further analysis of the published RNA-seq data about different maize tissues suggests that ZmWOX genes might be involved in nutrition and reproductive organ development (Figure 6).Two maize WOX genes, ZmWOX6 and 18, showed wide expression patterns in testing tissues, indicating their important role in maize development.Until now, four ZmWOX genes (e.g., ZmNS1, ZmNS2, ZmWUS1, and ZmWUS2, named in this work as ZmWOX3, 9, 2, and 22, respectively) have been reported.Double mutants of ZmNS1 and ZmNS2 caused maize leaves to become narrow, but single mutants displayed no obvious difference [36,49].This indicated the functional redundancy of ZmWOX3 and 9.Both two genes were highly expressed in inflorescence (Figure 6), implying that they may influence the reproductive process of maize.In Arabidopsis, the CLAVATA-WUSCHEL (CLV-WUS) model elaborates the regulation of division and differentiation of SAM [50].ZmWUS1 and 2 are homologous AtWUS gene but showed different expression patterns [37].Our study indicated ZmWUS1 (ZmWOX2) mainly expressed in the ear and inflorescence (Figure 6).At the same time, ZmWUS2 (ZmWOX22) showed high transcript levels in leaf tips (Figure 6), suggesting that ZmWUS1 and ZmWUS2 might be functionally differentiated.A recent study indicated that overexpression of TaWOX5 (homologous with AtWUS) in wheat could overcome the defects of genetic transformation relay on the genotype [51].The co-overexpression of ZmWUS2 and BBM genes in maize also improved genetic transformation efficiency [38].In addition to being responsible for growth and development, WOX genes also respond to abiotic stress [32].Except for ZmWOX4 and ZmWOX6, the other 20 WOX genes were positively modulated by waterlogging stress (Figure 7), suggesting that ZmWOXs may be involved in the morphological reconstruction of roots under hypoxic stress.Further RT-qPCR analysis indicated ZmWOX1, 4, 6, 13, 16, and 18 were positively or negatively modulated by abiotic stress (e.g., temperature, salt, and waterlogging) (Figure 8), indicating that ZmWOXs might also play a significant role in abiotic stress.
ZmWOX1 and ZmWOX18 were positively modulated by heat, cold, salt, and waterlogging stress (Figure 8).We further explored the localization and transactivation activity of ZmWOX1 and ZmWOX18.ZmWOX18 was localized in the nucleus, while ZmWOX1 was present in both the nucleus and cytoplasm (Figure 9A).ZmWOX18 had transactivation activity in yeast, but ZmWOX1 seemed unable to activate downstream genes (Figure 9B).Although ZmWOX1 and ZmWOX18 are positively regulated by abiotic stress signals, Zm-WOX1 and ZmWOX18 may play opposite roles in maize stress responses or even act reciprocally to obstruct the functioning of each other.However, this hypothesis needs to be further studied by using transgenic maize.Meanwhile, due to ZmWOX18 being induced by many abiotic stresses (Figure 8), and ZmWOX18 exhibited activating activity (Figure 9B).It might be a useful gene for agricultural practices for breeding stress-resilient maize varieties.Our findings may offer valuable information for future crop improvement strategies.

Plant Materials and Stress Treatments
A maize B73 inbred line was used in this work.Surface sterilized B73 seeds were planted in soil and grown under a 16/8 h day/night photoperiod at 25 • C in a greenhouse.V3-stage maize seedlings were used to assess abiotic stress.The seedlings were placed in a 42 • C or 4 • C growth chamber for either heat or cold stress for 0, 2, 4, or 8 h [52].The seedlings were transferred to a water solution containing 200 mM NaCl for 0, 2, 4, or 6 h for salt stress.The roots were collected for waterlogging by maintaining a 2 cm water layer above the first leaf for 0, 2, 4, or 6 h.There were three replicates for each treatment and three pots of seedlings for each replicate.The maize leaves (e.g., heat, cold, and salt) or roots (waterlogging) were harvested after stress treatments and then immediately frozen in liquid nitrogen and stored at −80 • C until use.

Chromosomal Localization and Collinearity Analysis
The chromosomal location information of the ZmWOXs was obtained from its genome annotation file in Maize GDB.Using MCScan (Version X, default parameters) [57] to explore the covariance relationships of WOX genes in different species, the result of chromosomal localization and collinearity analysis (using one-step MCScanX function) were then visualized by TBtools (v1.120) [56].

Transcriptome Data Analysis
The RNA-seq data used for tissue-specific expression analysis were obtained in a published report [40].The transcriptome sequencing data of maize under heat/cold [43], drought [41], waterlogging [44], and salt [42] stresses were used to explore the ZmWOX gene expression patterns under abiotic stress.All RNA-seq data were reanalyzed using the new maize B73 genome information (Section 4.2).Heatmap construction was made according to the fragments per kilobase per million (FPKM) values of sequencing data (2-3 biological replicates were plotted using mean values) and visualized by TBtools (v1.120) [56].The NCBI RNA-seq data links are shown in Supplementary Tables S4 and S5.

RT-qPCR Analysis of Gene Expression
The total leaf RNA extraction was performed following a published protocol [59].About 1 µg RNA was used for first-strand cDNA synthesis according to the description in the kit (CWBIO, Beijing, China).The RT-qPCR Mix (20 µL) included 6 µL diluted cDNA, 2 µL RNase-free water, a total of 2 µL each primer, and 10 µL SYBR Mix (Thermo Fisher Scientific, Waltham, MA, USA).PCR was performed on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) following this procedure: 94 • C for 5 min, followed by 43 cycles of 94 • C for 15 s and 60 • C for 25 s.Melting curves were obtained at 65-95 • C in the final step.There are three biological replicates (mixing three independent individuals for one biological replicate) for each sample and two technical replicates (calculation of mean values) for each biological replicate.The transcripts' relative abundance was calculated by the 2 −∆∆Ct method [60].The ZmActin1 gene (Zm00001eb348450) was used as the internal control.All primers are listed in Supplementary Table S6.

Subcellular Localization
The CDS of ZmWOX1 and ZmWOX18 were amplified using primers ZmWOX1-F/R and ZmWOX18-F/R (Supplementary Table S6) for subcellular localization of ZmWOX1 and ZmWOX18.They were then ligated into the pROKII vector [59].GFP (Empty pROKII vector) control or ZmWOX1/18-GFP vectors were separately cotransformed with a nuclear localization marker (D53-RFP) into 4-week-old tobacco epidermal cells following a published report [61].The cells were dark cultured for one day, under 16 h/8 h light/dark at 25 • C for two days.GFP, RFP, and chlorophyll autofluorescence signals (ChI) were observed with a scanning confocal microscope (Andor Revolution WD, Belfast, Northern Ireland, UK).

Statistical Analysis
Statistical analysis was performed using SPSS v19.0 (SPSS, Chicago, IL, USA).Significance was assessed using the Student's t-test.One or two asterisks against error bars of histograms are used to indicate means that are statistically different at p < 0.05 or p < 0.01, respectively."n" indicates the number of independent biological replicates.For RT-qPCR, the results are presented as the mean ± SD (n = 3 biological replicates, mixing three independent individuals for one biological replicate).

Conclusions
This study identified 22 ZmWOX genes, and a systematic analysis in maize was performed based on the last B73 genome data.All ZmWOX genes can be divided into three groups, and segmental duplication may be the main contribution to ZmWOX family expansion.Some WOX genes may be evolutionarily conservative in maize, wheat, sorghum, rice, and Arabidopsis.Published transcriptome data analysis showed that, except for ZmWOX6 and ZmWOX8, the expression of other ZmWOXs displayed tissue-specific patterns, suggesting that these genes may be involved in maize growth and development.Furthermore, six members of the ZmWOX genes, ZmWOX1, 4, 6, 13, 16, and 18, responded to the abiotic stresses.Meanwhile, ZmWOX1 and ZmWOX18 might play opposite regulatory roles in maize responses to stress.This study provides a reference for the subsequent ZmWOX gene function verification study.
MEME and TBtools were used to analyze the protein motifs and gene structure of ZmWOXs.According to the MEME-detected results, 10 conserved motifs were in Int.J. Mol.Sci.2024, 25, 441 5 of 19

Figure 2 .
Figure 2. Gene structure and conserved protein motifs of ZmWOXs.The clustering of ZmWOX genes was based on the phylogenetic tree shown in Figure 1.(A) Distribution of 10 conserved motifs (differently colored boxes) in ZmWOX proteins.The common sequences of motifs 1-10 are shown in Figure S1.(B) Exon-intron structure of 22 ZmWOXs.Introns as lines.

Figure 2 .
Figure 2. Gene structure and conserved protein motifs of ZmWOXs.The clustering of ZmWOX genes was based on the phylogenetic tree shown in Figure 1.(A) Distribution of 10 conserved motifs (differently colored boxes) in ZmWOX proteins.The common sequences of motifs 1-10 are shown in Figure S1.(B) Exon-intron structure of 22 ZmWOXs.Introns as lines.

Figure 3 .
Figure 3. Gene duplication events for ZmWOX genes in the maize genome.Schematic diagram of the relationship of ZmWOX genes between chromosomes.The chromosome locations of each ZmWOX gene are shown in the figure.The heatmap in the outside circles indicates chromosome gene density.The gray lines on the inside indicate collinear gene pairs in the maize genome.The collinear ZmWOX genes are connected through red line.

Figure 3 .
Figure 3. Gene duplication events for ZmWOX genes in the maize genome.Schematic diagram of the relationship of ZmWOX genes between chromosomes.The chromosome locations of each ZmWOX gene are shown in the figure.The heatmap in the outside circles indicates chromosome gene density.The gray lines on the inside indicate collinear gene pairs in the maize genome.The collinear ZmWOX genes are connected through red line.

Figure 4 .
Figure 4.The collinearity diagram of WOX genes between maize, wheat, sorghum, rice, and Arabidopsis.The gray line in the background represents the collinear gene pairs between the two species, and the WOX collinear genes are connected by red lines.

Figure 4 .
Figure 4.The collinearity diagram of WOX genes between maize, wheat, sorghum, rice, and Arabidopsis.The gray line in the background represents the collinear gene pairs between the two species, and the WOX collinear genes are connected by red lines.
ci. 2024, 25, x FOR PEER REVIEW 8 of 19 such as ABRE (contained in 20 members), MYB/MYC binding site elements related to abiotic stress and plant growth (contained in all members), and STRE elements (18 ZmWOXs) (Figure 5 and Supplementary Table

Figure 5 .
Figure 5.The cis-element analysis in promoter regions of ZmWOX genes.A promoter region of about 2 kb upstream of the ZmWOX genes was downloaded from the genome for analysis.Different colors represent different genes.The cis-element names and counts are listed in Supplementary TableS3.

Figure 6 .
Figure 6.The heatmap of ZmWOX gene expression levels in different maize tissue.The heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values of published RNA-seq data (Supplementary TableS4).The name of the tissues is labeled in the Figure.SAM: shoot apical meristem.

Figure 6 .
Figure 6.The heatmap of ZmWOX gene expression levels in different maize tissue.The heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values of published RNA-seq data (Supplementary TableS4).The name of the tissues is labeled in the Figure.SAM: shoot apical meristem.

Figure 7 .
Figure 7. Heatmap of ZmWOX gene expression levels under temperature, salt, waterlogging, and drought stresses.Different stresses are labeled in the figure.The data (except drought stress) represent the mean value of three replicates of published transcriptomic data (Supplementary TableS5).For salt stress, 'S' indicates a salt-sensitive maize-inbred line (L29).'T' indicates a salt-tolerant maizeinbred line (L87).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

Figure 7 .
Figure 7. Heatmap of ZmWOX gene expression levels under temperature, salt, waterlogging, and drought stresses.Different stresses are labeled in the figure.The data (except drought stress) represent the mean value of three replicates of published transcriptomic data (Supplementary TableS5).For salt stress, 'S' indicates a salt-sensitive maize-inbred line (L29).'T' indicates a salt-tolerant maize-inbred line (L87).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

Figure 9 .
Figure 9. Subcellular localization and transactivation activity assays of ZmWOX1 and ZmWOX18.(A) Subcellular localization of ZmWOX1 and ZmWOX18.The GFP or 35S∷ZmWOX1/18-GFP fusion expression vectors were cotransformed with mCherry (nuclear marker) into tobacco leaves using an Agrobacterium-mediated method.The GFP, mCherry, and chlorophyll autofluorescence (ChI) signals are labeled green, red, and purple, respectively.The empty GFP vector was used as a control.GFP: green fluorescent protein.Bars = 50 μm.(B) Transcription activation analysis of ZmWOX1 and ZmWOX18 in yeast.Full-length ZmWOX1 or ZmWOX18 was fused with the GAL4-DNA BD in the pGBKT7 vector (BD-WOX1 or BD-WOX18).BD-WOX1/18 and pGADT7(AD) were cotransformed into yeast Y2H gold.The yeast cells were cultured on a synthetic dextrose medium (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.

Author
Contributions: L.G. conceived research plans and designed experiments.X.C. and Y.H. conducted experiments.L.G. and X.C. wrote the draft.X.C., Y.H., Y.C. and B.W. analyzed the data.L.G. reviewed and edited this article.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Characteristics of WOX genes in maize.