JlA Teng-jiao, Ll Jing-jing, WANG Li-feng, CAO Yan-yong, MA Juan, WANG Hao, ZHANG Deng-feng, Ll Hui-yong

1 Institute of Cereal Crop, Henan Academy of Agricultural Sciences, Zhengzhou 450002, P.R.China

2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100089, P.R.China

Abstract The vacuolar proton-pumping pyrophosphatase gene (VPP) is often used to enhance plant drought tolerance via genetic engineering. In this study, the drought tolerance of four transgenic inbred maize lines overexpressing ZmVPP1 (PH4CV-T,PH6WC-T, Chang7-2-T, and Zheng58-T) and their transgenic hybrids was evaluated at various stages. Under normal and drought conditions, the height and fresh weight were greater for the four transgenic inbred maize lines than for the wild-type (WT) controls at the germination and seedling stages. Additionally, the transgenic plants exhibited enhanced photosynthetic efficiency at the seedling stage. In irrigated and non-irrigated fields, the four transgenic lines grew normally,but with increased ear weight and yield compared with the WT plants. Moreover, the ear weight and yield of the transgenic hybrids resulting from the PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W crosses increased in the non-irrigated field. Our results demonstrated that the growth and drought tolerance of four transgenic inbred maize lines with improved photosynthesis were enhanced by the overexpression of ZmVPP1. Moreover, the Chang7-2 and PH4CV transgenic lines may be useful for future genetic improvements of maize hybrids to increase drought tolerance.

Keywords: inbred maize lines, drought stress, ZmVPP1, photosynthesis

1. lntroduction

Crop production is limited by water scarcity worldwide(Pennisi 2008; Lobell et al. 2014). The future food demand for the rapidly increasing global population will likely aggravate the adverse effects of water deficits (Somerville and Briscoe 2001). Thus, many genes, including SbSNAC,GmDREB and AtNHX1, have recently been employed to improve the drought tolerance of crops via genetic engineering, which is a cost-effective and efficient method for genetic manipulations (Zhang et al. 2001; Farooq et al.2008; Mizoi et al. 2013; Mao et al. 2015). In Arabidopsis thaliana, vacuolar proton-pumping pyrophosphatase in A. thaliana (AtVPP) hydrolyzes cytosolic inorganic pyrophosphate (PPi) to form orthophosphate (Pi), with the released energy used to pump protons into vacuoles,thereby acidifying vacuoles and conserving energy in plants under drought conditions (Sarafian et al. 1992; Zhen et al.1997; Lin et al. 2012). Previous studies revealed that AtVPP expression contributes to plant growth and drought tolerance(Rea and Poole 1993; Gaxiola et al. 2001; Wehner et al.2015). Notably, overexpressing AtVPP in plants not only improves drought tolerance, but also enhances plant growth under normal conditions (Park et al. 2005; Pasapula et al.2011; Schilling et al. 2017).

There are several possible explanations for the phenotypes of transgenic plants carrying AtVPP (Schilling et al. 2017). For example, Shen et al. (2014) observed that transgenic cotton plants expressing the AtVVP1 and AtNHX1 genes maintain a higher photosynthetic rate than their wild-type (WT) counterparts under drought conditions.Wang et al. (2016) reported that the increased efficiency of photosynthesis and root development in transgenic maize plants carrying AtVPP enhances drought tolerance. However,the relationship between photosynthetic performance and drought tolerance in transgenic plants overexpressing VPP should be more thoroughly investigated. With the increased characterization of the relationships between fluorescence parameters and photosynthetic electron transport in vivo,analyses of fluorescence have been useful for studying photosynthetic performance when coupled with other noninvasive techniques such as gas analyses and infrared thermometry (Baker 2008; Suzuki et al. 2011).

Maize (Zea mays L.) is a major crop cultivated for food,feed and fuel because of its high yield and nutritional value.However, it is sensitive to drought stress and its yield is adversely affected by large fluctuations in weather patterns(Yu 2011; Boyer et al. 2013; Lobell et al. 2014). Although maize plants have recently been genetically modified to increase drought tolerance, the resulting transgenic inbred lines are difficult to use because of the complexity of the heterosis in maize (Ma et al. 2018). In the current study,transgenic inbred maize lines overexpressing Zea mays vacuolar proton-pumping pyrophosphatase (ZmVPP1) gene(Chang7-2-T, Zheng58-T, PH4CV-T, and PH6WC-T) and their hybrids were produced, after which their growth and drought tolerance were evaluated based on plant height,fresh weight, photosynthesis, ear weight, and yield.

2. Materials and methods

2.1. Transgenic inbred lines and hybrids

Transgenic inbred maize lines overexpressing ZmVPP1, their WT counterparts (PH4CV-W, PH6WC-W, Chang7-2-W, and Zheng58-W) and transgenic hybrids (PH4CV-T×PH6WC-W,Chang7-2-T×Zheng58-W, PH4CV-W×PH6WC-T, and Chang7-2-W×Zheng58-T) were produced with transgenic technology and hybridizations at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences(Beijing, China).

2.2. Total RNA extraction and analysis of ZmVPP1 expression

The TRIzol reagent (Sangon Biotech Co., Ltd., Shanghai,China) was used to extract total RNA from fresh leaves collected from the transgenic plants. The RNA was used as the template for synthesizing cDNA with the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Biomedical Technology Co., Ltd., Dalian, China) for a subsequent PCR-based analysis of ZmVPP1 expression. The maize GAPDH gene was used as an internal control to normalize the data. Primers specific for ZmVPP1 were designed with the Primer 5.0 Software. The PCR conditions were as follows:95°C for 5 min; 40 cycles of 95°C for 15 s, 60°C for 30 s,and 72°C for 30 s. The 2-ΔΔCtmethod (Livak and Schmittgen 2001) was used to quantify relative ZmVPP1 expression levels, and variations in expression was estimated by three biological replicates. The relative expression levels are presented herein as the mean±standard deviation (SD).

2.3. Screening for drought tolerance at the germination stage

Three replicates of seeds from the transgenic inbred lines and their WT controls were prepared at room temperature as follows. The seeds were surface-sterilized in 60 mL commercial Clorox bleach for 10 min, washed three times with distilled water, and placed on the upper third of nontoxic germination paper moistened with a captan solution(2.5 g L-1) (Zhongnong Hongtai Co., Ltd., Beijing, China).The seeds were positioned with the embryo facing down and the space was maximized to prevent contact between the developing root systems. The germination paper was then rolled vertically and placed into a plastic bucket containing 20% polyethylene glycol. The seed samples were then moved into a controlled-climate chamber set at 28°C and 45%relative humidity for a 3-day incubation in darkness and then a 4-day incubation with a 14-h day/10-h night photoperiod(light intensity of 5 000 lx). The resulting maize seedlings were harvested to measure the fresh weight and plant height.

2.4. Assessment of drought tolerance at the seedling stage

The seeds of the transgenic inbred lines and their WT controls were sterilized as described above and then placed in plastic containers (40 cm length×30 cm width×10 cm depth) filled with dried nutrient soil (2.5 kg) and a vermiculite mixture (0.5 kg). Sufficient water was added to the soil for a relative soil water content of 40%, after which the containers were placed in a greenhouse with a 14-h day/10-h night photoperiod and a temperature that ranged from (25±2) to (34±2)°C. At 15 days after sowing, drought conditions were simulated by completely withholding water until the soil moisture content decreased to 15%, after which it was maintained at 10-15% for 12 days by watering when necessary. The control maize seedlings received sufficient water to maintain a soil moisture content of 40-45%. The relative soil moisture content was monitored with the TDR 100 Soil Moisture Meter (Spectrum Technologies, Inc., IL, USA).

The drought-treated and control seedlings were analyzed regarding photosynthetic parameters (transpiration rate (Tr),net photosynthetic rate (Pn), stomatal conductance (Gs),and intercellular carbon dioxide concentration (Ci)) and fluorescence parameters (non-photochemical quenching(NPQ), photochemical quenching (qP) and electron transport rate (ETR)). Specifically, at 16 days after sowing, the seedlings were moved into a climate-controlled chamber and incubated in darkness for 20 min. The apical mature leaf was used for simultaneous and rapid measurements of the photosynthetic and fluorescence parameters with the GFS-3000 Portable Gas Exchange and Fluorescence System (Walz, Effeltrich, Germany). The system was set as follows: cuvette flow, 400 μmol s-1; cuvette temperature,28°C; actinic light, 930 μmol photons m-2s-1; measuring light pulse, modulated at 1 Hz; and saturating red light pulse,6 000 μmol m-2s-1at 10 Hz. Leaf basal fluorescence (Fo)was determined by measuring the light pulse, whereas the maximal fluorescence yield (Fm) was measured with saturating red pulses. The maximum quantum efficiency of photosystem II (Fv/Fm) was calculated as (Fm-Fo)/Fm.The maximum fluorescence yield (Fm′) and chlorophyll fluorescence yield (Fs) of the light-adapted leaves were measured by switching actinic light and saturating pulses at 60 s intervals for 20 min. Additionally, fluorescence of illuminated sample (Fo′) was calculated asFo/(Fv/Fm+Fo/Fm′)(Oxborough and Baker 1997). The quantum yield of the photosystem was calculated as (Fm′ -Fs)/Fm(Gentyet al.1989), whereasqPwas estimated as 1-(Fs-Fo′)/(Fm′-Fo′)(Bilger and Schreiber 1986). The ETR was estimated as (Fm′-Fs)/Fm×Photosynthetic active radiation×0.84×0.5(Maxwell and Johnson 2000). Moreover, NPQ was calculated as (Fm-Fm′)/Fm′.

2.5. Evaluation of the drought tolerance of transgenic inbred lines and hybrids under field conditions

For an analysis under field conditions, the seeds of transgenic inbred lines and their WT controls were planted in two-row plots under irrigated and non-irrigated conditions following a split plot design with three replicates at Yuanyang County, Henan Province, China (35°1′5′′N,113°42′10′′E). Drought stress was imposed at 30 days after sowing by stopping the irrigation until the plants growing in the non-irrigated field reached physiological maturity. The soil moisture content was quantified with the TDR 100 Soil Moisture Meter (Spectrum Technologies,Inc., IL, USA). The ears were harvested to measure the ear weight and yield.

2.6. Statistical analysis

Data were analyzed with the SPSS 20 Program (SPSS Inc., Chicago, IL, USA). Specifically, the data underwent a one-way analysis of variance andt-test, after which the least significant difference test was completed at the 95%confidence level (P＜0.05). Values are reported herein as the mean±SD.

3. Results

3.1. Relative ZmVPP1 expression in transgenic inbred maize lines

A total of 36 transgenic inbred lines carryingZmVPP1were produced, including 5 PH4CV-T, 10 PH6WC-T, 13 Chang7-2-T, and 8 Zheng58-T inbred lines (Table 1). Four transgenic inbred lines (Chang7-2-T, Zheng58-T, PH4CV-T,and PH6WC-T) that overexpressedZmVPP1relative to the corresponding expression level in their WT controls were selected for an evaluation of drought tolerance (Fig. 1).

3.2. Evaluation of the drought tolerance of transgenic inbred lines at the germination stage

Drought tolerance was investigated by treating samples with 20% polyethylene glycol. The simulated drought stress significantly inhibited the growth of the four transgenic inbred lines and their WT controls, resulting in significant decreases in plant height and fresh weight(P＜0.05) (Fig. 2). However, the fresh weights of Chang7-2-T, Zheng58-T, PH6WC-T, and PH4CV-T were greater than those of their WT controls under normal and droughtconditions, implying the transgenic plants were more drought-tolerant and grew better than their WT controls at the germination stage (Fig. 2-F).

Table 1 Acceptor of transgenic maize lines with ZmVPP1 gene

Fig. 1 Relative expression of Zea may vacuolar proton-pumping pyrophosphatase (ZmVPP1) gene in PH4CV-T, PH6WC-T,Chang7-2-T, and Zheng58-T transgenic inbred maize lines and their wild type (WT). Bars means SD (n=3).

Fig. 2 PH4CV-T (A), PH6WC-T (B), Chang7-2-T (C), and Zheng58 (D) inbred maize lines and their wild type (WT) and their plant heights (E) and fresh weights (F) at the germination stage under drying stress and control. W and T indicate WT and transgenic lines, respectively. Letters represent significant difference based on one-way analysis of variance (P＜0.05). Bar means SD (n=3).

3.3. lncreased drought tolerance and photosynthesis in the transgenic inbred lines at the seedling stage

At the seedling stage, the water deficit stress significantly restricted the growth of the four transgenic inbred lines and their WT controls, with significant decreases in plant height and fresh weight (Fig. 3). The fresh weight of PH4CV-T,PH6WC-T, Chang7-2-T, and Zheng58-T was 25.80, 5.51,182.07, and 41.27% higher than that of their WT controls,respectively, under drought conditions (Fig. 3-F; Table 2). An examination of photosynthetic and fluorescence parameters revealed that the leafPn,Tr,Gs,Ci,qP, and ETR of the four transgenic inbred lines and their WT controls decreased by varying degrees in response to drought stress (Fig. 4).Notably,Pnand ETR were higher in PH4CV-T, Chang7-2-T and Zheng 58-T than in the corresponding WT controls,which was indicative of effective photosynthesis (Fig. 4-B and H).

Fig. 3 Plant height (E) and fresh weight (F) of PH4CV-T (A), PH6WC-T (B), Chang7-2-T (C), and Zheng58-T (D) inbred lines and their wild type (WT) at the seedling stage under drying stress and control. W and T indicate WT and transgenic inbred lines,respectively. Letters represent significant difference based on one-way analysis of variance (P＜0.05). Bar means SD (n=3).

Table 2 Fresh weight (g) of transgenic inbred lines and their wild type (WT) at the seedling stage1)

The PH6WC-T transgenic line and its WT control were the most severely affected by the simulated drought stress among the analyzed transgenic inbred lines and controls, as indicated by the fact they had the lowestPnandTrand the highest NPQ (Fig. 4-E). Additionally, under normal conditions, the fresh weight,Pnand ETR of the four transgenic inbred lines were consistently higher than the corresponding values for the WT controls.

3.4. Ear weight of transgenic inbred maize lines and hybrids under field conditions

The four transgenic inbred maize lines and their hybrids were simultaneously planted in irrigated and non-irrigated fields. At 30 days after sowing, the soil moisture content decreased to 27.43% in the non-irrigated field and was maintained around 20% until harvest. The soil moisture content of the irrigated field was maintained at around 35%by irrigation (Fig. 5-E; Table 3). In the non-irrigated field,the exposure to constant drought conditions significantly delayed the onset of the silking stage in WT plants, but not in the transgenic inbred lines (Fig. 5-C). Additionally, the drought stress considerably decreased the ear weight of the transgenic inbred lines and their WT controls (Figs. 5-D and 6). However, the ear weight and yield were higher in the transgenic inbred lines than in their WT controls in both the non-irrigated and irrigated fields, which was indicative of the enhanced growth and drought tolerance of the transgenic plants.

Only two transgenic hybrids (PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W) had higher ear weights and yields compared with the control plants (PH4CV-W×PH6WC-W and Chang7-2-W×Zheng58-W, respectively) in both irrigated and non-irrigated fields (Fig. 7). These results suggested that the PH4CV-T and Chang7-2-T inbred lines may be useful for future studies involving the genetic improvement of maize hybrids.

Table 3 Soil moisture (%) of irrigated and no-irrigated field

Fig. 6 Ear weight of PH4CV-T (A and B), PH6WC-T (C and D), Chang7-2-T (E and F), Zheng58-T (G and H), and their wild type(WT) in irrigated and no-irrigated fields. W and T indicate WT and transgenic inbred lines, respectively. ＊, significant difference at P＜0.05. Bars mean SD (n=3).

4. Discussion

4.1. Overexpression of ZmVPP1

Improvements in the tolerance of maize to diverse biotic and abiotic stresses are one of the major factors that have increased maize yields (Duvick 2005). However, there has been an increase in the sensitivity of maize to drought stress associated with high vapor pressure deficits (Lobellet al.2014). Thus, enhancing drought tolerance has been a high priority for researchers involved in the genetic improvement of maize. The application of transgenic technology has resulted in the cloning of several genes for the genetic improvement of crops. TheVPPgene helps plants maintain a proton motive force in the cell and conserve energy by hydrolyzing PPi to form Pi in response to abiotic stress.

Fig. 7 Ear weight of hybrid of PH4CV-T×PH6WC-W (A and B) and Chang7-2-T×Zheng58-W (C and D) and their wild type (WT) in irrigated and no-irrigated fields. W and T indicate WT and transgenic inbred lines, respectively. ＊, significant difference at P＜0.05.Bars mean SD (n=3).

Therefore, the constitutive expression ofAtVPPcan increase the tolerance of plants to abiotic stresses (Yanget al. 2014; Khadilkaret al. 2016). Interestingly,AtVPPexpression also increases plant growth under non-stressed conditions (Schillinget al. 2017). Indeed, in the present study, the plant height, fresh weight, ear weight, and yield were greater for theZmVPP1-overexpressing transgenic inbred lines than for the WT controls under normal and drought conditions (Figs. 2, 3 and 6).

4.2. Overexpression of ZmVPP1 gene promoted photosynthesis of maize

Several studies have examined the phenotypes of transgenic plants carryingAtVPP, with a particular focus on the enhanced vacuolar ion regulation, increased auxin transport, greater heterotrophic growth, and increased transport of sucrose from the source to the sink tissues(Liet al. 2005; Yanget al. 2014; Khadikaret al. 2016).Additionally,VPPexpression was also observed to enhance photosynthesis and root development, with beneficial consequences for crop stress resistance (Shenet al. 2014;Wanget al. 2016). In the current study, we simultaneously monitored the gas exchange and fluorescence parameters of four transgenic inbred maize lines carryingZmVPP1at the seedling stage under normal and drought conditions.We revealed that the transgenic inbred lines maintained higherPn, ETR, andqP, but lower NPQ, than the WT controls,implying thatZmVPP1overexpression may protect the maize photosynthetic apparatus, thereby improving growth and drought tolerance. Future studies will need to clarify the effects of ZmVPP1on photosynthesis.

4.3. Utilization of transgenic inbred lines

Although many transgenic inbred maize lines exhibiting increased drought tolerance have been produced by the application of transgenic technology, few are used for the genetic improvement of hybrids because the heterosis in maize is complex (Maoet al. 2015; Wanget al. 2016;Maet al. 2018). We herein reveal that transgenic hybrids derived from the PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W crosses grow better and are more drought tolerant than the WT controls under both normal and drought conditions (Fig. 7). These findings imply that the Chang7-2-T and PH4CV-T inbred lines may be relevant for future genetic improvements of maize hybrids.

5. Conclusion

In this study, fourZmVPP1-overexpressingtransgenic inbred maize lines exhibited improved growth and drought tolerance as well as enhanced photosynthesis. Additionally,the crosses of the transgenic inbred lines (PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W) produced higher yielding hybrids under drought conditions.

Acknowledgements

This study was supported by the National Key Project for Research on Transgenic Plants, China (2016ZX08003-004)and the Independent Innovation Project of Henan Academy of Agricultural Sciences, China (2060302).