*4.2. Reverse Transcription PCR and RT-qPCR Analysis*

Total RNA was isolated from the collected leaf samples using the TRIZOL reagent (Biotopped, China) according to the manufacturer's instructions. One microgram of the total RNA from each sample was used in reverse transcription. RT-qPCR analyses were performed in the optical 48-well plates, using the ABI7300 Thermo-cycler (Applied Biosystems, USA). Reactions were carried out in a

10 µL volume containing 1 µL diluted cDNA, 200 nM gene-specific primers, and 5 µL SYBR Premix Ex Taq II (Takara, China) with the following conditions—10 min at 95 ◦C, 40 cycles of 15 s at 95 ◦C, and 30 s at 60 ◦C. Each experiment was performed using at least three independent biological replicates. *ZmTCP32* and *ZmTCP42* RNA levels in response to different stresses were calculated according to a standard curve calibration, based on amplification of a dilution series of *ZmTCP32* and *ZmTCP42* plasmid, respectively, according to the manufacturer's protocol. *ZmUbi-2* (UniProtKB/TrEMBL; ACC: Q42415) was used as the internal control. The expression levels of different stress-responsive genes were compared, based on the delta Ct method with normalization to *ACTIN2*.

#### *4.3. Generation of Transgenic Plants*

The coding region of the *ZmTCP42* cDNA of the maize B73 inbred line was inserted into the pGreenII vector [58]. The constructed plasmid carrying the desired gene was transformed into *Agrobacterium tumefaciens* GV3101 + pSoup and transformed into ecotype Col-0, as described previously [58]. Using the kanamycin-based selection, several independent T<sup>2</sup> transgenic lines were obtained, and expression of the *ZmTCP42* transgene was confirmed in these lines by RT-PCR and RT-qPCR. Two independent overexpression lines *ZmTCP42-OE16* and *ZmTCP42-OE25* (with a single inserted copy) were selected, based on the level of transgene expression, and the T3 homozygous seeds were subjected to further analyses.

#### *4.4. Seed Germination and Drought Phenotype Analysis*

To study the effect of ABA on germination and cotyledon greening, the seeds were planted on MS medium plates, with an addition of plus 1% sucrose and different concentrations of ABA (Sigma-Aldrich)in a growth chamber, at 22 ◦C under a 16-h-light/8-h-dark photo-period, with a 60% relative humidity, after 2 days of vernalization in darkness at 4 ◦C. For the drought stress tolerance test, plants were grown for 28 days under normal conditions and subjected to water stress by withholding watering for 14 days [58]. Four plants were planted in each small cup with 100 g soil (1:1 of black soil/vermiculite)and grown under the condition of 16-h-light/8-h-dark. Four independent experiments were performed and at least 16 plants each were observed, for the *ZmTCP42-OE16* and *ZmTCP42-OE25* lines and the WT, in each experiment. The plants were rewatered when significant differences in wilting were observed. Three days after rewatering, the surviving plants were counted.

## *4.5. Identification of the TCP Proteins in the Maize Genome*

To identify TCP proteins in the B73 maize genome, some putative TCP protein sequences from maize (genome assembly: B73\_RefGen\_v3) were first retrieved from the Plant Transcription Factor Database 3.0 (Jin et al. 2014). Conserved TCP DNA-binding domain (PF03634) from the Pfam database [59] was used to search and retrieve sequences from the Phytozome database v10.0 (available online: http://www.phytozome.net/eucalyptus.php). In addition, BLASTP searches were also performed against the maize genome, to identify any additional TCP members using *Arabidopsis* and rice TCP proteins sequences (*E-value* ≤ 0.00001). The overlapped genes were removed. The presence of a TCP domain in all family proteins was evaluated using the CDD database searches (https://www.ncbi.nlm.nih.gov/Structure/cdd). Arabidopsis TCP proteins were downloaded from TAIR 10 (available online: http://www.arabidopsis.org), which contained 24 members. TCP proteins in rice (Genome assembly: Rice Genome Annotation Project Database release 7) and sorghum (Genome assembly: V3) were both downloaded from the Plant Transcription Factor Database. The ExPASy program (http://www.expasy.org/tools/) was used to predict the molecular weight (kDa) and the isoelectric point (PI) of each protein.

#### *4.6. Gene Structure and Phylogenetic Analysis*

Genomic sequences of maize TCP genes were downloaded from Phytozome V10, and the untranslated regions were removed. To show the exon/intron organization for individual TCP genes, coding sequences were aligned to genomic sequences and schematics generated using GSDS 2.0 (Gene Structure Display Server) (available online: http://gsds.cbi.pku.edu.cn) [60]. To determine the phylogenetic relationships of the TCP proteins, full-length amino acid sequences of TCPs identified in maize, rice, *Arabidopsis*, and sorghum were aligned by the ClustalW program [61]. The Phylogenetic tree was constructed by the neighbor-joining method, with 1000 bootstrap replicates.
