*2.2. Maize Contains Roughly Twice as Many TCP Genes as Rice and Sorghum*

In order to study the phylogenetic relationships among the predicted maize TCPs, we constructed a neighbor-joining tree for the ZmTCP proteins and their orthologs from rice, sorghum and *Arabidopsis*, based on multiple-alignment of the full-length TCP protein sequences (Figure 1). As shown on the phylogenetic tree, the 113 TCPs were classified into two main classes—class I and class II. Class II was further divided into two clades—CYC/TB1 and CIN. The phylogenetic tree based on the sequence alignments of the 46 ZmTCPs was also divided into three clades (Figure S1). The boundaries of these major clades were clearly stated by the phylogenetic locations of several canonical *TCP* genes, such as the class I genes *OsPCF1* and *OsPCF2*, the *CIN*-like class II genes *AtTCP2*, *AtTCP3*, and *AtTCP4*, and the *CYC*/*tb1*-like class II genes *AtTCP1*, *AtTCP12*, *AtTCP18*, and *ZmTCP2*/*ZmTB1*. In agreement with previous work, all *Arabidopsis*, rice, and sorghum TCPs fell in the same class or clade, as previously reported in our phylogenetic tree [11,12]. Interestingly, dicot *Arabidopsis* TCP proteins clustered separately from those of the three monocot plants in the same clad, and some proteins from three of the monocot plants displayed pairing. Furthermore, maize and sorghum TCPs were found to share a closer phylogenetic relationship than maize and rice ones, which was consistent with the notion that sorghum is a closer relative of maize than rice. Throughout the phylogenetic tree, there were 49 TCPs in the PCF clade, 34 in CIN, and 30 in CYC/TB1 (Figure 1). Among the 46 *ZmTCPs*, there were 17 *ZmTCPs* in class I and 29 *ZmTCPs* in class II; within class II, 10 in CIN and 19 in CYC/TB1 (Figure S1). Significantly, the number of maize *ZmTCPs* was roughly twice as large as the number in each of rice and sorghum. Specifically, among all 30 CYC/TB1 *TCP* genes, 19 were from maize, 4 from sorghum, and 3 each from rice and *Arabidopsis* (Figure 1). This result suggests that the *ZmTCP* gene family in the allopolyploid maize genome underwent a two-fold duplication. This expansion was biased and occurred mainly in the class II CYC/TB1 clade.


**Table 1.** Detailed information for 46 *ZmTCP* genes in the *Zea mays* L. genome.

duplication. This expansion was biased and occurred mainly in the class II CYC/TB1 clade.

*ZmTCP36* GRMZM2G089638 3 1251 416 1 42.91 5.79 PCF *ZmTCP37* GRMZM2G092214 3 975 324 2 34.35 6.7 PCF *ZmTCP38* GRMZM2G359599 4 522 173 4 19.58 8.6 CYC/TB1 *ZmTCP39* GRMZM2G170232 4 396 131 4 14.38 8.87 CYC/TB1 *ZmTCP40* GRMZM2G178603 5 663 220 1 22.76 10.02 PCF *ZmTCP41* GRMZM2G077755 5 1200 400 1 40.49 9.2 PCF *ZmTCP42* GRMZM2G180568 7 972 323 1 33.71 8.11 CIN *ZmTCP43* GRMZM2G148022 8 2337 778 15 84.78 7.35 CIN *ZmTCP44* GRMZM2G034638 8 948 315 2 33.08 6.57 PCF *ZmTCP45* GRMZM2G424261 10 489 162 3 17.27 5.91 CIN *ZmTCP46* GRMZM2G093895 10 1218 405 1 41.09 5.95 PCF

In order to study the phylogenetic relationships among the predicted maize TCPs, we constructed a neighbor-joining tree for the ZmTCP proteins and their orthologs from rice, sorghum and *Arabidopsis*, based on multiple-alignment of the full-length TCP protein sequences (Figure 1). As shown on the phylogenetic tree, the 113 TCPs were classified into two main classes—class I and class II. Class II was further divided into two clades—CYC/TB1 and CIN. The phylogenetic tree based on the sequence alignments of the 46 ZmTCPs was also divided into three clades (Figure S1). The boundaries of these major clades were clearly stated by the phylogenetic locations of several canonical *TCP* genes, such as the class I genes *OsPCF1* and *OsPCF2*, the *CIN*-like class II genes *AtTCP2*, *AtTCP3*, and *AtTCP4*, and the *CYC/tb1*-like class II genes *AtTCP1*, *AtTCP12*, *AtTCP18*, and *ZmTCP2/ZmTB1*. In agreement with previous work, all *Arabidopsis*, rice, and sorghum TCPs fell in the same class or clade, as previously reported in our phylogenetic tree [11,12]. Interestingly, dicot *Arabidopsis* TCP proteins clustered separately from those of the three monocot plants in the same clad, and some proteins from three of the monocot plants displayed pairing. Furthermore, maize and sorghum TCPs were found to share a closer phylogenetic relationship than maize and rice ones, which was consistent with the notion that sorghum is a closer relative of maize than rice. Throughout the phylogenetic tree, there were 49 TCPs in the PCF clade, 34 in CIN, and 30 in CYC/TB1 (Figure 1). Among the 46 *ZmTCPs*, there were 17 *ZmTCPs* in class I and 29 *ZmTCPs* in class II; within class II, 10 in CIN and 19 in CYC/TB1 (Figure S1). Significantly, the number of maize *ZmTCPs* was roughly twice as large as the number in each of rice and sorghum. Specifically, among all 30 CYC/TB1 *TCP* genes, 19 were from maize, 4 from sorghum, and 3 each from rice and *Arabidopsis* (Figure 1). This result

*2.2. Maize Contains Roughly Twice as Many TCP Genes as Rice and Sorghum* 

**Figure 1.** Phylogenetic tree of the predicted Teosinte-branched 1/Cycloidea/Proliferating (TCP)proteins from maize, rice, sorghum, and *Arabidopsis*. The phylogenetic tree was constructed based on the sequence alignment of the 113 full-length TCP protein sequences from four species. The unrooted tree was drawn by MEGA 7.0 with the neighbor-joining (NJ)method, using the following parameters—bootstrap values (1000 replicates)and the poisson model. The scale refers to the branch lengths. The gene codes and names are illustrated in red for maize, black for rice, blue for sorghum, and green for *Arabidopsis*. The names used for rice and the Arabidopsis *TCP* genes are from a previous report [8].

## *2.3. Many TCP Genes are Found in the Syntenic Segments of Rice, Sorghum, and Maize Genomes*

As gene synteny is indicative of the homologous gene function, we explored the collinearity of rice, sorghum, and maize *TCP* genes. We collected gene collinearity data from the Plant Genome Duplication Database (PGDD, http://chibba.agtec.uga.edu/duplication, Table S1)using *ZmTCP* genes as anchors, then defined each genomic syntenic block as the chromosomal segment consisting of multiple homologous genes, across species. According to this analysis, about 20 *ZmTCP* genes were found to have syntenic members or collinear genes in rice and sorghum, as shown in Figure 2. Some chromosomal segments containing *ZmTCP* genes, including *ZmTCP2*, *3*, *8*, 9, *19, 25*, *29*, *40*, and *ZmTCP41*, were found to have been evolutionally conserved between maize, rice, and sorghum (Figure 2). This indicated that not only the individual genes but these entire chromosomal segments were evolutionally conserved. Two segments on maize chromosomes 2 and 7, containing a duplicated gene pair *ZmTCP5* and *ZmTCP23,* share synteny with two segments on rice chromosomes 7 and 9, carrying the corresponding genes *OsTCP22* and *OsTCP24*. Two other segments on maize chromosomes 2 and 4, containing another duplicated gene pair *ZmTCP33* and *ZmTCP16,* share synteny with two segments on rice chromosomes 11 and 12, carrying corresponding genes *OsPCF3* and *OsTCP28*. Interestingly, these two paralogous gene pairs, *ZmTCP5*/*ZmTCP23* and *ZmTCP16*/*ZmTCP33*, share the same syntenic blocks on sorghum chromosomes 2 and 7, respectively, indicating that these duplicated gene pairs might have arisen from segmental duplication in maize, after maize and sorghum diverged evolutionarily (Figure 2). One rice chromosomal block, containing *OsTCP19*, shares synteny with two segments on maize chromosomes 6 and 9, with a duplicated gene pair *ZmTCP21* and *ZmTCP26.* However, only one syntenic block could be found in the sorghum genome. Another fragment on rice chromosome 1 containing *OsTCP6* and *OsTCP5* is also duplicated on maize chromosomes 3 and 8, carrying two duplicated gene pairs *ZmTCP10*/*43* and *ZmTCP37*/*44.* More interestingly, their orthologs in

sorghum are also tandemly duplicated on only a single chromosomal segment. Additionally, one maize chromosomal block containing *ZmTCP32* shares synteny with two segments on rice chromosomes 8 and 9, containing its orthologous genes *OsPCF2* and *OsTCP25*; *SbTCP* orthologs could not be found, although the syntenic segment in sorghum was identified. Based on these data, we concluded that most of the *TCP* genes existed before the species diverged, but some *ZmTCP* genes might have originated from later segmental duplication or accompanied the generation of an allotetraploid maize genome. *Int. J. Mol. Sci.* **2019**, *20*, x 6 of 18

**Figure 2.** Schematic diagram of syntenic chromosomal segments containing *ZmTCP* genes between the rice, sorghum, and maize genomes. The maize, rice, and sorghum chromosomes are abbreviated Zm, Os, Sb, respectively. Homologous chromosomal regions between the different genomes are linked by black dotted lines and pale blue shaded regions. Each *TCP* orthologous gene pair was connected by a red line. Yellow boxes indicate homologous segments between the maize and the sorghum genomes, while the brown boxes identify the homologous regions in the maize and rice **Figure 2.** Schematic diagram of syntenic chromosomal segments containing *ZmTCP* genes between the rice, sorghum, and maize genomes. The maize, rice, and sorghum chromosomes are abbreviated Zm, Os, Sb, respectively. Homologous chromosomal regions between the different genomes are linked by black dotted lines and pale blue shaded regions. Each *TCP* orthologous gene pair was connected by a red line. Yellow boxes indicate homologous segments between the maize and the sorghum genomes, while the brown boxes identify the homologous regions in the maize and rice genomes.

#### genomes. *2.4. Expression Profiles of ZmTCP Genes*

maize seedlings.

*2.4. Expression Profiles of ZmTCP Genes*  According to previous studies, *TCP* genes had key roles in different aspects of plant development, as well as in response to stress [8,37]. In order to gain further insights into the roles of the *ZmTCP* genes, we assessed their tissue-specific expression profiles from the available transcriptomic data of maize B73 [43]. An expression heatmap was constructed for the 46 *ZmTCPs* in different tissues, from 15 developmental stages, under non-limiting growth conditions (Figure 3A). Results indicated that the expression patterns of different *ZmTCP* genes varied greatly. Transcripts of *ZmTCP37* and *ZmTCP44* showed relatively high levels of expression, compared to the other *ZmTCPs* examined. It is noteworthy that *ZmTCP2/TB1* and *ZmTCP18,* a homolog of *OsTB1*, were expressed relatively high in cobs and husk leaves, while *ZmTCP25* was expressed relatively high in endosperm and mature seeds. Additionally, in the B73 variety grown under non-limiting conditions, According to previous studies, *TCP* genes had key roles in different aspects of plant development, as well as in response to stress [8,37]. In order to gain further insights into the roles of the *ZmTCP* genes, we assessed their tissue-specific expression profiles from the available transcriptomic data of maize B73 [43]. An expression heatmap was constructed for the 46 *ZmTCPs* in different tissues, from 15 developmental stages, under non-limiting growth conditions (Figure 3A). Results indicated that the expression patterns of different *ZmTCP* genes varied greatly. Transcripts of *ZmTCP37* and *ZmTCP44* showed relatively high levels of expression, compared to the other *ZmTCPs* examined. It is noteworthy that *ZmTCP2*/*TB1* and *ZmTCP18,* a homolog of *OsTB1*, were expressed relatively high in cobs and husk leaves, while *ZmTCP25* was expressed relatively high in endosperm and mature seeds. Additionally, in the B73 variety grown under non-limiting conditions, all *ZmTCP* genes in class I (PCF clade)were expressed relatively high in different tissues.

Next, we investigated the expression profiles of all maize *TCP* genes, in response to drought stress, using microarray analysis. Based on their expression patterns, the maize *TCP* genes could mainly be classified into four groups (Figure 3B). The expression levels of 14 *ZmTCP* genes were continuously up-regulated (fold-change > 1) in response to both drought stress conditions (5 h and 10 h of drought treatments). Among these genes, *ZmTCP1*, *ZmTCP9*, and *ZmTCP24* showed two-fold changes (or more) in one or two drought stress conditions. In contrast, 19 *TCP* genes were continuously down-regulated (fold-change < −1) under both drought stress conditions, of which

all *ZmTCP* genes in class I (PCF clade) were expressed relatively high in different tissues.

results clearly showed the functional divergence of *ZmTCP* genes in response to drought stress, in

*Int. J. Mol. Sci.* **2019**, *20*, x 7 of 18

**Figure 3.** Expression patterns of *ZmTCP* genes in tissues and in response to drought conditions. (**A**) A heat map depicting gene expression levels of 46 *ZmTCPs* in fifteen different tissues from various developmental stages. Normalized gene expression values are shown in different colors that represent the levels of expression indicated by the scale bar. The gray color represents unavailable data. (**B**) Microarray-based expression analysis of *ZmTCP* genes. A heat map was generated based on the fold-change values in the treated samples, when compared with the unstressed control. The color scale for fold-change values is shown at the bottom. The drought-treated leaf samples were collected **Figure 3.** Expression patterns of *ZmTCP* genes in tissues and in response to drought conditions. (**A**) A heat map depicting gene expression levels of 46 *ZmTCPs* in fifteen different tissues from various developmental stages. Normalized gene expression values are shown in different colors that represent the levels of expression indicated by the scale bar. The gray color represents unavailable data. (**B**) Microarray-based expression analysis of *ZmTCP* genes. A heat map was generated based on the fold-change values in the treated samples, when compared with the unstressed control. The color scale for fold-change values is shown at the bottom. The drought-treated leaf samples were collected at two time points, 5 and 10 h, which reflected relative leaf water content (RLWC)of 70% and 60%, respectively.

at two time points, 5 and 10 h, which reflected relative leaf water content (RLWC) of 70% and 60%,

respectively. *2.5. Association Analysis of Natural Variations in ZmTCP Genes Identified Two ZmTCP Genes Associated with Drought Tolerance in Maize*  In order to further investigate whether the natural variations in any of the ZmTCP TFs are associated with the different drought tolerance levels of maize varieties, we conducted an association analysis for these genes. The drought tolerance level of each inbred line was investigated by evaluating its survival rate under severe drought stress, at the seedling stage. To assess potential associations between survival rates and *ZmTCPs*, we utilized previously reported methods and data [44,45] and previously identified single nucleotide polymorphism (SNP) markers, to characterize the Next, we investigated the expression profiles of all maize *TCP* genes, in response to drought stress, using microarray analysis. Based on their expression patterns, the maize *TCP* genes could mainly be classified into four groups (Figure 3B). The expression levels of 14 *ZmTCP* genes were continuously up-regulated (fold-change > 1)in response to both drought stress conditions (5 h and 10 h of drought treatments). Among these genes, *ZmTCP1*, *ZmTCP9*, and *ZmTCP24* showed two-fold changes (or more)in one or two drought stress conditions. In contrast, 19 *TCP* genes were continuously down-regulated (fold-change < −1)under both drought stress conditions, of which *ZmTCP19* showed about four fold change in one or two drought stress conditions. Expression of the remaining 13 genes, were either suppressed or induced under one of the drought conditions. The results clearly showed the functional divergence of *ZmTCP* genes in response to drought stress, in maize seedlings.

#### presence of genetic polymorphisms in each of these 46 *ZmTCP* genes. Among the 46 identified *ZmTCP* genes, 26 were found to be polymorphic with an average of 12 SNPs (Table 2), while the polymorphic information of the other 16 genes was currently absent (Minor Allele Frequency, MAF *2.5. Association Analysis of Natural Variations in ZmTCP Genes Identified Two ZmTCP Genes Associated with Drought Tolerance in Maize*

≥ 0.05). **Table 2.** Association analysis of the natural variation in *ZmTCP* genes with respect to drought tolerance at the seedling stage in the maize diversity panel. **Locus ID Gene Name Polymorphic Name GLM PCA PCA + K**  *p* **≤ 0.01** *p* **≤ 0.01** *p* **≤ 0.01** *p* **≤ 0.001** In order to further investigate whether the natural variations in any of the ZmTCP TFs are associated with the different drought tolerance levels of maize varieties, we conducted an association analysis for these genes. The drought tolerance level of each inbred line was investigated by evaluating its survival rate under severe drought stress, at the seedling stage. To assess potential associations between survival rates and *ZmTCPs*, we utilized previously reported methods and data [44,45] and previously identified single nucleotide polymorphism (SNP) markers, to characterize the presence of

GRMZM2G166687 *ZmTCP1* - - - - - AC233950.1\_FG002 *ZmTCP2* 3 2 0 0 0 GRMZM2G115516 *ZmTCP3* 33 0 0 0 0 genetic polymorphisms in each of these 46 *ZmTCP* genes. Among the 46 identified *ZmTCP* genes, 26 were found to be polymorphic with an average of 12 SNPs (Table 2), while the polymorphic information of the other 16 genes was currently absent (Minor Allele Frequency, MAF ≥ 0.05).


**Table 2.** Association analysis of the natural variation in *ZmTCP* genes with respect to drought tolerance at the seedling stage in the maize diversity panel.

*ZmTCP37* was found to be the most polymorphic, with 34 SNPs in this natural diversity panel. Subsequently, three statistical models were applied to identify significant genotypic and phenotypic associations. Specifically, a general linear model (GLM)with the first two principal components (PC2)and a mixed linear model (MLM)were used to find associations (Figure 4A). The GLM method was applied to perform single-marker analysis. Then PC2, via the first two principal components of the SNP data, was applied to correct for spurious associations caused by the population structure. The MLM method, incorporating both PC<sup>2</sup> and a Kinship matrix (to correct for the effect of cryptic

relatedness), was considered to be effective for controlling false positives in the association analysis (Figure 4A) [46,47]. The candidate gene association analysis detected significant associations between the genetic variations of *ZmTCP32* and *ZmTCP42* and drought tolerance, under different models, with a *p*-value ≤ 0.01 (Table 2; Figure 4B,C). However, under the standard mixed linear model (MLM), the only two significantly associated SNPs contributing to the phenotype of drought tolerance were both located at the 50 UTR region of *ZmTCP42*, which suggested that this candidate gene was significantly associated with drought tolerance (*p*-value ≤ 0.001, −log10*p* = 3.77) (Figure 4C). We further analyzed the survival rates of maize inbred lines carrying *ZmTCP42* drought-tolerant or drought-sensitive alleles. It was found that *ZmTCP42AA* was a favorable drought tolerance allele (Figure S2). The two-fold induction of *ZmTCP42* expression by dehydration (Figure 3B)suggested that the RNA level of *ZmTCP42* was likely more correlated with drought tolerance than other potential variations among different *Int. J. Mol. Sci.*  maize varieties. **2019**, *20*, x 9 of 18

**Figure 4.** Association analysis of genetic variations in *ZmTCP32 and ZmTCP42* with maize drought tolerance. (**A**) Quantile–quantile plots of the estimated –log10(*p*) from the *ZmTCP* gene family-based association analysis, using three methods. The gray line is the expected line under the null distribution. The white square represents the observed *p* values using general linear model (GLM); the gray square represents the GLM model with the first two principal components (PC2); the black diamond represents the observed *p* values using the mixed linear model (MLM) model incorporating both PC2 and a Kinship matrix. (**B**,**C**) Schematic diagrams of *ZmTCP32* (**B**) and *ZmTCP42* (**C**), including the UTR (gray), intron (thin black line), and protein coding regions (thick black line), are presented in the x-axis. The *p* value is shown on a –log10 scale. **Figure 4.** Association analysis of genetic variations in *ZmTCP32 and ZmTCP42* with maize drought tolerance. (**A**) Quantile–quantile plots of the estimated −log10(*p*) from the *ZmTCP* gene family-based association analysis, using three methods. The gray line is the expected line under the null distribution. The white square represents the observed *p* values using general linear model (GLM); the gray square represents the GLM model with the first two principal components (PC<sup>2</sup> ); the black diamond represents the observed *p* values using the mixed linear model (MLM) model incorporating both PC<sup>2</sup> and a Kinship matrix. (**B**,**C**) Schematic diagrams of *ZmTCP32* (**B**) and *ZmTCP42* (**C**), including the UTR (gray), intron (thin black line), and protein coding regions (thick black line), are presented in the x-axis. The *p* value is shown on a −log10 scale.

#### *2.6. ZmTCP32 and ZmTCP42 are both Induced by ABA Treatment and Drought Stress 2.6. ZmTCP32 and ZmTCP42 Are Both Induced by ABA Treatment and Drought Stress*

treatment (+3.0-fold at 24 h) (Figure 5).

To confirm whether *ZmTCP32* and *ZmTCP42* RNA levels are truly associated with drought tolerance, we used RT-qPCR to directly analyze the RNA expression of the *ZmTCP32* and *ZmTCP42* genes, in response to ABA treatment and drought stress. As illustrated in Figure 5, in the B73 genotype, *ZmTCP32* and *ZmTCP42* are both significantly induced by ABA by roughly four-fold, relative to the controls at 48 h after the ABA treatment. More importantly, *ZmTCP42* was highly induced by drought stress (+7.6-fold at 24 h) and by the PEG6000 treatment (+5.9-fold at 24 h); *ZmTCP42* was significantly more responsive to dehydration stress and PEG treatment than *ZmTCP32*, even though *ZmTCP32* was also responsive to drought stress (+3.9-fold at 24 h) and PEG To confirm whether *ZmTCP32* and *ZmTCP42* RNA levels are truly associated with drought tolerance, we used RT-qPCR to directly analyze the RNA expression of the *ZmTCP32* and *ZmTCP42* genes, in response to ABA treatment and drought stress. As illustrated in Figure 5, in the B73 genotype, *ZmTCP32* and *ZmTCP42* are both significantly induced by ABA by roughly four-fold, relative to the controls at 48 h after the ABA treatment. More importantly, *ZmTCP42* was highly induced by drought stress (+7.6-fold at 24 h) and by the PEG6000 treatment (+5.9-fold at 24 h); *ZmTCP42* was significantly more responsive to dehydration stress and PEG treatment than *ZmTCP32*, even though *ZmTCP32* was also responsive to drought stress (+3.9-fold at 24 h) and PEG treatment (+3.0-fold at 24 h) (Figure 5).

Arabidopsis.

**Figure 5.** The RNA levels of *ZmTCP32* and *ZmTCP42* in maize B73 leaves measured by RT-qPCR, after treatment with 100  μM ABA, drought stress, or 10% PEG treatment. The drought stress was performed as previously described [45], on hydroponically cultured seedlings, for 5 and 24 h, and their relative leaf water contents (RLWC) were determined to be approximately 70% and 58%, at the corresponding time points, respectively. *ZmUbi-2* transcript levels were used as an internal control for data normalization; the gene-specific primers are listed in Table S2. Each data point represents the mean ± SD (*n* = 3) of three biological replicates. Significant differences were calculated by one-way ANOVA with Duncan's multiple test (SAS Institute, Inc., Cary, NC, USA). Different letters indicate a significant statistical difference between sample means, at *p* = 0.05, while means with the same letters were not significantly different. **Figure 5.** The RNA levels of *ZmTCP32* and *ZmTCP42* in maize B73 leaves measured by RT-qPCR, after treatment with 100 µM ABA, drought stress, or 10% PEG treatment. The drought stress was performed as previously described [45], on hydroponically cultured seedlings, for 5 and 24 h, and their relative leaf water contents (RLWC) were determined to be approximately 70% and 58%, at the corresponding time points, respectively. *ZmUbi-2* transcript levels were used as an internal control for data normalization; the gene-specific primers are listed in Table S2. Each data point represents the mean ± SD (*n* = 3) of three biological replicates. Significant differences were calculated by one-way ANOVA with Duncan's multiple test (SAS Institute, Inc., Cary, NC, USA). Different letters indicate a significant statistical difference between sample means, at *p* = 0.05, while means with the same letters were not significantly different.

#### *2.7. Overexpression of ZmTCP42 Enhances Drought Resistance in Transgenic Arabidopsis*

*2.7. Overexpression of ZmTCP42 Enhances Drought Resistance in Transgenic Arabidopsis*  Given that the polymorphism in the 5′-UTR of *ZmTCP42* suggests a potential role in drought resistance on maize seedlings, we selected *ZmTCP42* to directly test for its function. To validate *ZmTCP42* function in response to drought stress, we generated transgenic Arabidopsis plants that overexpress the *ZmTCP42* gene, by using the enhanced cauliflower mosaic virus 35S promoter. After screening for the *ZmTCP42* expression levels, we selected two independent transgenic lines, *ZmTCP42-OE16* and *ZmTCP42-OE25*, with enhanced RNA levels, for further experiments (Figure 6A,B). We investigated the ABA sensitivity of *ZmTCP42* transgenic lines, in response to exogenous ABA, during seed germination. In the absence of exogenous ABA, all lines germinated completely, as did the wild-type seeds; on the contrary, in the presence of exogenous ABA, the expanding, greening cotyledons were significantly affected in the *ZmTCP42*-OE lines; the number of seedlings with green cotyledons in *ZmTCP42-OE16* and *ZmTCP42-OE25* were significantly lower than those of the wild-type at 1 μM ABA (Figure 6C,D), suggesting that the *ZmTCP42* overexpression lines are more sensitive to ABA. Furthermore, we analyzed the tolerance levels of *ZmTCP42*-OE lines to drought stress. When the 28-day-old transgenic seedlings were subjected to the drought test, only ~30% of the wide-type plants were able to recover from the stress, whereas ~65 of *ZmTCP42-OE16*  and ~87% of *ZmTCP42-OE25* transgenic plants survived (Figure 6E, F). Similar results were obtained in repeated experiments, indicating that overexpression of *ZmTCP42* enhanced drought tolerance in Given that the polymorphism in the 50 -UTR of *ZmTCP42* suggests a potential role in drought resistance on maize seedlings, we selected *ZmTCP42* to directly test for its function. To validate *ZmTCP42* function in response to drought stress, we generated transgenic Arabidopsis plants that overexpress the *ZmTCP42* gene, by using the enhanced cauliflower mosaic virus 35S promoter. After screening for the *ZmTCP42* expression levels, we selected two independent transgenic lines, *ZmTCP42-OE16* and *ZmTCP42-OE25*, with enhanced RNA levels, for further experiments (Figure 6A,B). We investigated the ABA sensitivity of *ZmTCP42* transgenic lines, in response to exogenous ABA, during seed germination. In the absence of exogenous ABA, all lines germinated completely, as did the wild-type seeds; on the contrary, in the presence of exogenous ABA, the expanding, greening cotyledons were significantly affected in the *ZmTCP42*-OE lines; the number of seedlings with green cotyledons in *ZmTCP42-OE16* and *ZmTCP42-OE25* were significantly lower than those of the wild-type at 1 µM ABA (Figure 6C,D), suggesting that the *ZmTCP42* overexpression lines are more sensitive to ABA. Furthermore, we analyzed the tolerance levels of *ZmTCP42*-OE lines to drought stress. When the 28-day-old transgenic seedlings were subjected to the drought test, only ~30% of the wide-type plants were able to recover from the stress, whereas ~65 of *ZmTCP42-OE16* and ~87% of *ZmTCP42-OE25* transgenic plants survived (Figure 6E,F). Similar results were obtained in repeated experiments, indicating that overexpression of *ZmTCP42* enhanced drought tolerance in Arabidopsis.

We further assessed the expression levels of ABA- inducible or drought-inducible genes by using real-time RT-qPCR to analyze their responses in *Arabidopsis*. Upon dehydration stress, the RNA levels of *RAB18*, *RD29A*, *LEA14*, and *RD17*, which are well-known drought-responsive, positive regulator genes of drought tolerance, were up-regulated, relative to wild-type *Arabidopsis* plants (Figure 6G).

Similar changes were observed for the RNA levels of the *RbohD* and *RbohF* genes, encoding two NADPH oxidases, which were directly responsible for the reactive oxygen species (ROS) production in leaves, in response to stress (Figure 6G). These results clearly showed that *ZmTCP42* 

**Figure 6.** Analysis of wild-type (WT) and *ZmTCP42-*overexpressing transgenic *Arabidopsis* plants. (**A**) RT-PCR analysis of *ZmTCP42* transcript levels in *ZmTCP42* transgenic lines. (**B**) RT-qPCR analysis of *ZmTCP42* transcript levels in *ZmTCP42* transgenic lines. (**C**) Seed germination of the *ZmTCP42* transgenic lines and in the wild-type, in response to abscisic acid (ABA). Germination rates with 1  μM ABA for 5 days, defined by cotyledon greening, in wild-type *ZmTCP42-OE16* and *ZmTCP42- OE25*, compared to the MS medium alone. (**D**) Statistical analysis of a green seedling's rate of *ZmTCP42* transgenic lines and wild-type grown on Murashige and Skoog (MS) medium with 0, 0.5  μM, and 1  μM ABA. (**E**) Drought tolerance of the *ZmTCP42* transgenic Arabidopsis plants. Photographs were taken before and after the drought treatment, followed by 3 days of rewatering. (**F**) Statistical analysis of survival rates after the drought-stress treatment. The average survival rates and standard errors were calculated from three independent experiments. (**G**) RNA levels of stressresponsive genes in the *ZmTCP42* transgenic lines and in the wild-type, in response to drought stress. Total RNA was obtained from 3 weeks-old seedlings treated by 3 h of dehydration stress and was analyzed by RT-qPCR, using the gene-specific primers listed in Table S2. The mean value and standard error were calculated from three replicates after normalization to *ACTIN2*. The RNA levels in the wild-type grown under non-stress conditions was taken as 1.0. Significant differences were calculated by one-way ANOVA with Duncan's multiple test (SAS Institute, Inc., Cary, NC, USA). **Figure 6.** Analysis of wild-type (WT) and *ZmTCP42-*overexpressing transgenic *Arabidopsis* plants. (**A**) RT-PCR analysis of *ZmTCP42* transcript levels in *ZmTCP42* transgenic lines. (**B**) RT-qPCR analysis of *ZmTCP42* transcript levels in *ZmTCP42* transgenic lines. (**C**) Seed germination of the *ZmTCP42* transgenic lines and in the wild-type, in response to abscisic acid (ABA). Germination rates with 1 µM ABA for 5 days, defined by cotyledon greening, in wild-type *ZmTCP42-OE16* and *ZmTCP42-OE25*, compared to the MS medium alone. (**D**) Statistical analysis of a green seedling's rate of *ZmTCP42* transgenic lines and wild-type grown on Murashige and Skoog (MS) medium with 0, 0.5 µM, and 1 µM ABA. (**E**) Drought tolerance of the *ZmTCP42* transgenic Arabidopsis plants. Photographs were taken before and after the drought treatment, followed by 3 days of rewatering. (**F**) Statistical analysis of survival rates after the drought-stress treatment. The average survival rates and standard errors were calculated from three independent experiments. (**G**) RNA levels of stress-responsive genes in the *ZmTCP42* transgenic lines and in the wild-type, in response to drought stress. Total RNA was obtained from 3 weeks-old seedlings treated by 3 h of dehydration stress and was analyzed by RT-qPCR, using the gene-specific primers listed in Table S2. The mean value and standard error were calculated from three replicates after normalization to *ACTIN2*. The RNA levels in the wild-type grown under non-stress conditions was taken as 1.0. Significant differences were calculated by one-way ANOVA with Duncan's multiple test (SAS Institute, Inc., Cary, NC, USA). Different letters indicate a significant statistical difference between the sample means at *p* = 0.05, while means with the same letters were not significantly different.

We further assessed the expression levels of ABA- inducible or drought-inducible genes by using real-time RT-qPCR to analyze their responses in *Arabidopsis*. Upon dehydration stress, the RNA levels of *RAB18*, *RD29A*, *LEA14*, and *RD17*, which are well-known drought-responsive, positive regulator genes of drought tolerance, were up-regulated, relative to wild-type *Arabidopsis* plants (Figure 6G). Similar changes were observed for the RNA levels of the *RbohD* and *RbohF* genes, encoding two NADPH oxidases, which were directly responsible for the reactive oxygen species (ROS) production in leaves, in response to stress (Figure 6G). These results clearly showed that *ZmTCP42* overexpression improved the inducibility of *Arabidopsis* drought-tolerance-associated genes upon drought stress, leading to an enhanced drought tolerance.

#### **3. Discussion**

To date, the TCP family members have been described in various species; for instance, 24 *TCP* genes in *Arabidopsis*, 23 in rice, 20 in sorghum, and 66 in wheat (*Triticum aestivum* L.) [11,12,16,17]. Chai et al. (2017)reported 29 *ZmTCP* genes in maize, including their chromosomal location, structure and domain conservation analysis, and gene duplication analysis. They also analyzed the phylogenetic inference and expression profiling of these 29 *ZmTCP* genes, and then speculated that the *ZmTCP* genes influence stem and ear growth. In our study, we retrieved a total of 46 *ZmTCP* genes, by searching against the updated maize genome B73\_RefGen\_v3, including the 29 *ZmTCPs* previously reported by Chai et al. and the 17 newly identified *ZmTCPs* containing a canonical TCP domain. Subsequently, we further systematically determined their phylogenetic relationship, synteny with rice, sorghum, and Arabidopsis *TCPs*, pattern of drought-responsiveness, association analysis of their natural variations with drought tolerance, and functional analysis of *ZmTCP42* in drought tolerance. Collectively, our data demonstrated that a few *ZmTCP* family proteins are likely involved in plant drought tolerance; in particular, ZmTCP42 functions as an important positive regulator for drought tolerance.

Recently, genome-wide identification revealed that segmental duplication might be the main contributor to the expansion of the *TCP* gene family in some monocots, including rice, sorghum and wheat, as well as in some dicot species, such as cotton and soybean [14,17,48–52]. The report showed that there were about 2.6–2.8-fold duplication of the *TCP* gene family in allotetraploid upland cotton genome (*G. hirsutum*), compared to *Arabidopsis* [48]. Similarly, we found that the duplication ratio of the *ZmTCP* family was about 2-fold in the allopolyploid maize genome, compared with *Arabidopsis*, rice, and sorghum. The change in the ratio of gene numbers suggests that the *ZmTCP* family has undergone lineage-specific expansion and functional divergence during the course of evolution. Furthermore, as shown in the phylogenetic analysis, monocot ZmTCP proteins clustered independently from those in *Arabidopsis*, suggesting a potential functional divegence between the dicot and monocot TCPs. We also found that *ZmTCP* gene expansion was not uniform, and occurred mainly in the class II CYC/TB1 clade. However, the biological significance of the *ZmTCP* gene duplication in the maize genome remains to be determined. Our collinearity analysis on *TCP* genes within several monocot plants showed that 20 *ZmTCP* genes had syntenic members or collinear genes in rice and sorghum, and that the chromosomal segments containing these genes were also duplicated (Figure 2), supporting the concept that the maize genome might have arisen from an ancestral allotetraploid, half of which share a common ancestor with sorghum, which in turn probably represents a lineage split from rice [53,54]. Taken together, our results might provide some useful clues for future studies on a homologous gene function.

A growing body of research suggest that TCP TFs play important roles in plant development, as well as in response to abiotic stress [8,19,33,34]. However, the role of *TCP* genes in plant response to drought stress in maize was still obscure, without answers to which, the *ZmTCP* genes were directly associated with the levels of drought tolerance in maize. We were, therefore, prompted to perform this study to address this question. The answer would not only help facilitate the genetic improvement of drought tolerance but would also increase our knowledge of the biological function of this gene family. Generally, *ZmTCP* genes exhibit great differential expression in response to drought stress in maize seedlings, not only among subgroups but members within the same subgroup, suggesting that these *ZmTCP* genes might function very diversely. Our current results showed that 14 *ZmTCP* genes were significantly up-regulated and 19 genes were clearly down-regulated, indicating that these genes might function as key mediators of drought stress responses in maize (Figure 3). Notably, three genes, *ZmTCP1*, *ZmTCP9*, and *ZmTCP24*, belonging to the class II CYC/TB1 clade, showed similar inducible expression patterns (Figure 3), implying that they might play a redundant role in regulating drought stress response in maize. Moreover, the orthologs of *ZmTCP9* and *ZmTCP24* in rice and *Arabidopsis* are *OsPCF5* and *AtTCP3*/*AtTCP4*, respectively, which are regulated by the conserved miR319. Previous reports have shown that knockdown of miR319-dependent TCPs (by constitutively overexpressing miR319)increased drought and salinity tolerance [34,55]. The rice homolog of *ZmTCP1* and *ZmTCP13* was named *OsTB1*, which plays an important role in stress response, especially in regulation of cold tolerance [55,56]. Additionally, *ZmTCP37* and *ZmTCP44*, orthologs of *AtTCP20*, also showed a slightly inducible expression pattern under drought stress (Figure 3). In *Arabidopsis*, *AtTCP20* represses the transcription of *LIPOXYGENASE2* (*AtLOX2*) gene, which is involved in jasmonic acid synthesis and promotes leaf senescence [57].

To explore the association of *TCPs* with stress tolerance in plants, we have investigated homologous *ZmTCP* genes in a small number of inbred lines and proposed predictive conclusions. Among the 46 *ZmTCP* genes analyzed, the genetic polymorphisms of *ZmTCP32* and *ZmTCP42* were significantly associated with the phenotypic variations of drought tolerance (*p*-value ≤ 0.01, PC2) (Table 2). While *ZmTCP42* was the most significantly (*p*-value ≤ 0.001, MLM) associated with drought tolerance in this natural variation panel, the natural variation in the *ZmTCP42* promoter might contribute to maize drought tolerance (Figure 4). In the B73 genotype of maize, *ZmTCP32* was constitutively highly expressed in various tissues (Figure 3A). In comparison with *ZmTCP32*, *ZmTCP42* RNA was detected at a low level in various tissues, except in leaves and endosperm, and showed a relatively high level at 10 h, upon drought stress in a micrroarry analysis (Figure 3B). Both *ZmTCP32* and *ZmTCP42* were significantly induced by ABA, dehydration and PEG treatment; more notably, *ZmTCP42* was induced higher than *ZmTCP32* in response to dehydration and PEG treatment. These results suggested that *ZmTCP42* was involved in plant drought response. Transgenic *Arabidopsis* overexpressing *ZmTCP42* exhibited a higher ABA sensitivity, improved drought stress tolerance, and enhanced the induction of Arabidopsis ABA- or drought-inducible genes, which strongly suggests that ZmTCP42 might regulate these ABA/drought-response genes. In summary, we have identified ZmTCP42 as an important positive regulator of drought tolerance, through analyses of gene expression and natural variations.
