Informatics analysis of ZmHDZIV13
The full-length cDNA sequence of ZmHDZIV13 was cloned from the inbred line Zheng 58 by RT-PCR, resulting in a length of 2097 bp. The full-length DNA sequence of the gene (GenBank accession number: BK008038) was obtained by searching the National Center for Biotechnology Information database with the cDNA sequence as a probe. Sequence alignment showed that the gene contained 10 exons and 9 introns. The DNA sequence of ZmHDZIV13 was used as a probe to search through a maize genome database (https://maizegdb.org/). We found that the ZmHDZIV13 gene was located near the telomere on chromosome 4. Protparam analysis showed that the ZmHDZIV13 gene, with a predicted molecular weight of 7.62 kDa and an isoelectric point of 6.19, encoded 698 amino acids. CD-Search was used to analyze the conserved domain of the ZmHDZIV13 protein. The results showed that the ZmHDZIV13 protein had two conserved domains, HD and START, which was consistent with that of the HDG protein (Fig. 1).
Transformation of ZmHDZIV13 into Arabidopsis
The results of the transformation of Agrobacterium showed that the plant expression vector pCAMBIA3300-35S-ZmHDZIV13-bar was successfully introduced into the strain LBA4404. The transgenic line 35S::ZmHDZIV13 was screened by adding 300 μg/L basta in the medium. We obtained 53 homozygous lines of ZmHDZIV13 in the T3 generation. Expression analysis of ZmHDZIV13 resulted in 39 resistant plants with specific bands amplified near the 2097-bp size, while no corresponding bands were found in the untransformed plants. The PCR-positive transformation rate of resistant plants was 81.25%, indicating that the exogenous gene was successfully integrated into the Arabidopsis genome. The amplification results are shown in Figure 2 (homozygous lines L3, L7, L25).
Morphological and physiological characteristics in response to drought stress
After seven days of drought treatment, the survival rate and leaf RWC of the T3 generation of overexpression and WT plants were determined. The results showed that the survival rates of transgenic (L3, L7, L25) and WT plants were 100% when given sufficient water. In contrast, the survival rates of WT, L3, L7 and L25 grown in the drought stress condition decreased by 82.0%, 57.63%, 64.41% and 68.38% of that of the WT control, respectively. The survival rates of L3, L7 and L25 were 135.39%, 97.72% and 75.67% higher than those of the WT under stress, respectively. Where water availability was sufficient, the RWCs of WT, L3, L7 and L25 remained above 91%, and the differences among these groups were not obvious. With the temporary drought, the RWCs of leaves of L3, L7 and L25 were reduced. Moreover, RWC of the WT reached the lowest amount, 59.09%, while the RWCs of the ZmHDZIV13-transgenic plants basically remained above 50% and were 70.89% (L3), 66.88% (L7) and 30.40% (L25) higher than that of the WT (Fig. 3). The results showed that the water holding capacity of the T3 generation of plants that overexpressed ZmHDZIV13 was stronger than that of WT plants subjected to the temporary drought.
The contents of MDA and free proline are important indexes used to reflect drought resistance of crops. Proline is an osmoprotectant that reduces the osmotic potential of cells in response to drought stress. MDA is a product of plant cell membrane lipid peroxidation. Drought stress causes plant cells to lose water and eventually damages the membrane system observed in the ruptures of the vacuolar membrane and damages to the lipid membrane structure. In our experiment, the contents of MDA and proline were determined from WT, L3, L7 and L25 plants exposed to the drought stress. The results showed that the contents of MDA and proline in the non-transformed and transformed lines were similar to the corresponding contents in the regularly irrigated group. After seven days of water deprivation, the MDA contents of L3, L7 and L25 decreased significantly by 13.67%, 15.63% and 13.60%, respectively, and the proline contents of L3, L7 and L25 increased significantly by 10.14%, 9.74% and 4.99%, respectively, from that of their non-deprived groups (Fig. 3). The results showed that the physiological indexes related to drought resistance observed from the transformed plant leaves were improved in response to the temporary drought treatment.
Morphological characteristics in response to different ABA levels
ABA signaling stimulates plant responses to various stress factors by regulating the expression of stress- and ABA-responsive genes and the closure of leaf stomata . In order to determine whether the ZmHDZIV13 gene improves drought resistance in transgenic Arabidopsis through the ABA signaling pathway, the phenotypes of WT and transgenic A. thaliana seedlings grown on MS medium containing 0.6 μM ABA were observed and compared. There were no significant differences in seedling and root lengths between WT and the transgenic L3, L7 and L25 plants grown in MS medium without ABA (Fig. 4). In the medium containing 0.6 μM ABA, the growth of WT and transgenic Arabidopsis seedlings was significantly inhibited, and the degree of inhibition was significantly greater in transgenic than in WT plants. These results indicate that overexpression of ZmHDZIV13 can significantly increase the sensitivity of transgenic plants to ABA.
Morphological characteristics in response to NaCl treatments and osmotic stress in Arabidopsis
The WT Arabidopsis and 35S::ZmHDZIV13 transgenic lines (L3, L7, and L25) were subjected to osmotic stress tests on a medium supplemented with NaCl and mannitol to determine potential interactive effects of genetics and environmental conditions. The results showed that the germination rate of transgenic plants increased with the increase of NaCl concentrations, but the germination rate of transgenic plants was lower than that of WT plants (Fig. 5). These results indicate that ZmHDZIV13 can improve germination and cotyledon-emergence rates of transgenic plants under osmotic and salt stresses.
Root length, number of lateral roots and root dry weight of 35S::ZmHDZIV13 and WT Arabidopsis seedlings under NaCl and mannitol stresses were measured or counted. The number of lateral roots from ZmHDZIV13 was higher than that from WT (Figs. 5 and 6). Transgenic 35S::ZmHDZIV13 seedlings showed strong osmotic resistance under high (150 mM) NaCl/mannitol stress. With the addition of 150 mM NaCl, the average root length and root dry weight of transgenic Arabidopsis seedlings decreased by 57.59% and 4.59%, respectively, while the number of lateral roots increased by 10.34%. The root length, root dry weight and number of lateral roots of WT decreased by 80.95%, 35.23% and 67.47%, respectively. Due to treatment with 150 mM mannitol, the average root length and root dry weight of transgenic Arabidopsis seedlings decreased by 11.97% and 11.36%, respectively, while the number of lateral roots increased by 88.50%. The root length, root dry weight and number of lateral roots of WT decreased by 11.94%, 54.55% and 72.29%, respectively (Fig. 6). The results showed that 35S::ZmHDZIV13 seedlings exhibited stronger tolerance to the drought stress, and it might be due to the increase of number of lateral roots.
Relative expression levels of stress response genes under drought stress
In order to elucidate the molecular mechanism of drought tolerance of the ZmHDZIV13 gene, the transcription levels of six drought-related genes were compared by quantitative PCR. The results showed that ABA and drought-stress-inducible genes (P5CS1, RD22, RD29B, RD29A, ERD1, and NCED3) were highly expressed in dehydrated plants, and the expression levels of drought genes in transgenic plants were significantly higher than that in WT plants (Fig. 7). For example, the relative expression levels of RD29B in ZmHDZIV13 transgenic lines L3, L7 and L25 were respectively 127.70, 126.41 and 153.05 at the 6th day of dehydration. In addition, we found that drought could induce the expression of ERD1, but there was no significant difference in expression between transgenic and WT Arabidopsis. Before the drought treatment, with the exception of RD29B, the expression levels of all other genes in transgenic plants were lower than those in WT.
Leaf density and water-use efficiency in transgenic tobacco
Heterologous reproduction experiments were carried out with transgenic tobacco, the genetic structure pCAMBIA3300-35S-ZmHDZIV13-bar was transferred into tobacco and further studied using three independent over expression lines T2 homozygous lines (L7, L10 and L17) (Fig. 8). The results showed that stomatal density of transgenic ZmHDZIV13-overexpression lines in L10 and L17, but not in L7, were lower than that in WT lines (Fig. 8A and E), while the size of a single stomate of each transgenic line was greater than that of WT lines (Fig. 8F). The lower stomatal density may be related to the greater size of epidermal cells (Fig. 8B), and the decrease of stomatal density may help to reduce water loss from the leaves of transgenic plants. The photosynthetic rates were significantly greater, transpiration rates were significantly lower (Fig. 8B and C), and WUEs were significantly higher in transgenic than in WT lines (Fig. 8D).