Abstract
Key message
ZmPIF3 plays an important role in ABA-mediated regulation of stomatal closure in the control of water loss, and can improve both drought tolerance and did not affect the grain yield in the transgenic rice.
Abstract
Phytochrome-interacting factors (PIFs) are a subfamily of basic helix-loop-helix (bHLH) transcription factors and play important roles in regulating plant growth and development. In our previous study, overexpression of a maize PIFs family gene, ZmPIF3, improved drought tolerance in transgenic rice. In this study, measurement of water loss rate, transpiration rate, stomatal conductance, guard cell aperture, density and length of ZmPIF3 transgenic plants showed that ZmPIF3 can enhance water-saving and drought-resistance by decreasing stomatal aperture and reducing transpiration in both transgenic rice and transgenic Arabidopsis. Scrutiny of sensitivity to ABA showed that ZmPIF3 transgenic rice was hypersensitive to ABA, while the endogenous ABA level was not significantly changed. These results indicate that ZmPIF3 plays a major role in the ABA signaling pathway. In addition, DGE results further suggest that ZmPIF3 participates in the ABA signaling pathway and regulates stomatal aperture in rice. Comparison analysis of the phenotype, physiology, and transcriptome of ZmPIF3 transgenic rice compared to control plants further suggests that ZmPIF3 is a positive regulator of ABA signaling and enhances water-saving and drought-resistance traits by reducing stomatal openings to control water loss. Moreover, investigation of the agronomic traits of ZmPIF3 transgenic rice from four cultivating seasons showed that ZmPIF3 expression increased the tiller and panicle number and did not affect the grain yield in the transgenic rice. These results demonstrate that ZmPIF3 is a promising candidate gene in the transgenic breeding of water-saving and drought-resistant rice plants and crop improvement.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Water scarcity greatly affects plant growth and development and reduces crop yield. Under a water deficit, plants activate a diverse set of physiological, metabolic, and defense systems to survive and sustain growth, such as reducing water losses by adjusting stomatal movement (opening and closing) (Sirichandra et al. 2009). The majority of stomatal movement occurs through stomatal pores. Stomatal pores located in the epidermis of plant leaves control the uptake of CO2 for photosynthesis and water loss during transpiration (Hetherington and Woodward 2003; Huang et al. 2009; Murata et al. 2015). Plant transpiration rates can be regulated by stomatal movement. Thus, stomatal pores are the primary defense mechanism to prevent water loss under drought stress and play a crucial role in drought tolerance.
The plant stress hormone abscisic acid (ABA) can induce stomatal closure to maintain the water status in plant cells under water deficit conditions (Cutler et al. 2010; Murata et al. 2015). ABA is involved in the transcriptional and post-transcriptional regulation of some stress-responsive genes (Chinnusamy et al. 2004). Upon drought treatment, the ABA concentration quickly elevates to promote the binding of ABA to the receptors PYR/PYL/RCAR, which inhibits type 2C (PP2C) protein phosphatases and may result in release of SNF1-related type 2 protein kinases (SnRK2s) from the inhibition of PP2C protein (Ma et al. 2009; Yoshida et al. 2014). The activated SnRK2s can phosphorylate downstream ion channels and transcription factors that can bind the ABA response element and regulate the expression of ABA-responsive genes (Lee and Luan 2012; Boursiac et al. 2013). The regulation of several ion channels at the plasma membrane and tonoplast are related with ABA-induced stomatal closure. Therefore, ABA is a key mediator in drought stress response because it induces stomatal closing to minimize transpirational water loss (Cutler et al. 2010; Kim et al. 2010; Osakabe et al. 2014).
Phytochrome-interacting factors (PIFs) are a subset of basic helix-loop-helix (bHLH) transcription factors (Castillon et al. 2007; Leivar and Quail 2011). PIFs were first described as central players in transducing light signals perceived by the light sensing phytochrome (phy) photoreceptors (Castillon et al. 2007; Bae and Choi 2008; Leivar and Quail 2011; Leivar and Monte 2014). It has been reported that phytochrome B (phyB), the upstream regulator of PIFs, were able to reduce water loss and improve drought tolerance by regulating stomatal opening, density and stomatal length. Stomata of phyB cop1 mutants opened less widely than those of cop1 mutants, and stomata of the pif3 pif4 mutants opened wider than those of the wild-type, indicating that COP1, together with PIFs, may act downstream of phyB in regulating stomatal opening (Wang et al. 2010). The rice deficient in phyB exhibited reduced stomatal density and stomatal length, which presumably results in a reduction of water loss and transpiration rate and thus enhanced drought tolerance (Liu et al. 2012). A further study found that phyB enhances drought tolerance by increasing stomatal sensitivity to ABA. PhyB mutants were less sensitive than the wild-type in diminishing stomatal conductance in response to exogenous ABA application (Staneloni et al. 2008; Boccalandro et al. 2009; Gonzalez et al. 2012).
Moreover, a recent study revealed that the PIFs-like protein OsPIL1/OsPIL13, acting as a key regulator of reduced internode elongation, was down-regulated under drought stress conditions in rice (Todaka et al. 2012). The rice PIF gene OsPIF14 expression was shown to be modulated by drought (Cordeiro et al. 2016). It has also been reported that double overexpression of OsPIL1 and DREB1A can improve drought tolerance in Arabidopsis (Kudo et al. 2016). In our previous studies, we cloned two maize PIF genes, ZmPIF1 and ZmPIF3, and found that these genes regulate plant response to drought (Gao et al. 2015, 2018). The solid evidence shows that ZmPIF3 is involved in drought stress responses and has potential use for improving drought stress tolerance (Gao et al. 2015). Understanding the role of ZmPIF3 in response to drought stress is a prerequisite for genetic improvement of drought stress tolerance. In this study, we further study the role of ZmPIF3 in drought stress and suggest the maize ZmPIF3 protein can play an important role in an ABA-associated drought stress response by controlling stomatal closure.
Materials and methods
Plant materials and stress treatments
Rice (Oryza sativa L. Wuyunjing) for transgenic analysis was grown in a controlled environment chamber at 28/25 °C with a 16/8 h photoperiod and 70% relative humidity. Plasmid construction and rice transformation with ZmPIF3 was conducted as stated in Gao et al. (2015). Three homozygous transgenic lines (OE3, OE5, OE11) with higher expression levels of the target gene were selected for phenotypic assays, and all seeds used for phenotypic assays were from the same harvest and stored under the same conditions. Wild-type Arabidopsis thaliana (Columbia 0 type) and homozygous transgenic Arabidopsis were placed in a climate chamber at 22 °C with 70% relative humidity and a 12/12 h photoperiod. All tests in this study were repeated a minimum of three times.
Phenotype of transpiration assay
Forty-day-old wild-type, vector-only control and transgenic rice seedlings were planted in transparent pots filled with the same volume of hydroponic culture solution. There were 35 rice seedlings planted in each pot. Seedlings were cultured in a greenhouse without watering for 3 days. After 3 days, the water levels in the pots containing ZmPIF3 transgenic and control rice were marked with black lines.
Thirty-day-old seedlings of WT (col) and T3 ZmPIF3 transgenic Arabidopsis were transplanted into the same transparent pots (containing the same weight of soil), and 300 ml water was added after saturation of the soil with water. After 4 days, the water levels in the pots containing the ZmPIF3 transgenic and WT Arabidopsis were marked with black lines. The remaining water in the WT and ZmPIF3 transgenic Arabidopsis was measured after 4 days.
Measurements of water loss rate, transpiration rate, and stomatal conductance
Water-loss rates of detached leaves from ZmPIF3 transgenic lines and wild-type plants were measured by monitoring the fresh weight loss at indicated time points. This assay was performed at room temperature (~ 23 °C) with 35% relative humidity. Water loss was calculated as the percentage of initial fresh weight at each time point. Transpiration rates and stomatal conductance were measured using a portable photosynthesis system (Li-Cor 6400; Li-Cor, Lincoln, NE, USA) in the morning (9–11 AM). Three measurements were made for each plant, and ten plants were used for both the wild-type and the transgenic plants. All parameters were measured in 40-day-old well-watered plants in a greenhouse.
Observations of leaf stomata by scanning electron microscopy (SEM)
Flag leaves of 40-day-old rice and leaves of 30-day-old Arabidopsis were detached from the plants of ZmPIF3 transgenic lines and WT. The samples were immediately fixed with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS; pH 6.8) to avoid any alterations. The stomatal pictures were obtained using an environmental scanning electron microscope (XL-30ESEM, Philips). Stomata were counted at random in eight visual sections on the abaxial epidermis, and final tallies were used to compute their densities. Length and apertures were measured randomly from a minimum of 140 stomata on the same specimens, using Image-Pro Plus6.0 software (Media Cybernetics, Singapore).
Germination assay and growth measurement
For testing the ABA sensitivity of transgenic plants at the germination stage, 35 surface-sterilized seeds were subsequently sown on sterile filter papers in square boxes supplemented with 0, 5, 10 µM ABA after 1 day of gemination. The germination rate of the treated seeds was calculated after 5 day. The filter papers were divided into four parts with black lines to sow the ZmPIF3 transgenic lines and wild-type seeds, respectively.
To measure seedling plant height, 20 surface-sterilized seeds were transplanted into 96-well plates which were moved to the bottom of the plate after the shoot reached 2 cm in height. Seeds were grown in water containing 0, 5 µM ABA. After 7 days of growth, plant height was measured. To measure seedling root growth, 20 surface-sterilized seeds were vertically grown in water containing 0, 10 µM ABA. After 5 days of growth, root length was measured.
Quantification of the endogenous ABA content
Measurement of endogenous ABA levels of flag leaves was performed. 40-day-old seedlings of the ZmPIF3 transgenic lines and the wild-type plants were used for ABA quantification. Briefly, rice leaves were ground in liquid nitrogen, homogenized in 1 ml distilled water, and then shaken at 4 °C overnight (~ 12 h). The homogenates were centrifuged at 12,000×g for 10 min at 4 °C, and the supernatant was used directly for ABA assays. ABA analysis was performed using the radioimmunoassay method (Zhang et al. 2015). The 450 µl reaction mixture contained 200 µl phosphate buffer (pH 6.0), 100 µl 1:5000 diluted antibody (Mac 252; Abcam, Cambridge, MA, USA) solution, 100 µl [3H] ABA (~ 8000 c.p.m.; Sigma-Aldrich) solution, and 50 µl crude extract. The mixture was then incubated at 4 °C for 90 min, and the bound radio activity was measured in 50% saturated (NH4)2SO4-precipitated pellets with a liquid scintillation counter (Beckman LS6500; Becton Dickinson, Franklin Lakes, NJ, USA).
DGE analysis
Flag leaves of 40-day-old ZmPIF3 transgenic lines (OE3, OE5 and OE11) and wild-type seedlings (30 plants each) were harvested from a greenhouse, snap frozen immediately in nitrogen, and stored at − 80 °C until further processing. Three independent replicates were collected from each individual line. Total RNAs were extracted from the samples using RNAprep pure Plant Kit (Tiangen, China) according to the manufacturer’s instructions. The process of digital gene expression (DGE) was conducted according to the standard protocol of the Beijing Genomics Institute (http://www.genomics.cn/index; Shenzhen, China).
Grain yield analysis
To evaluate yield components of transgenic plants under normal field conditions, four independent T3 (2014), T4 (2015), T5 (2016) and T6 (2017) homozygous lines of the ZmPIF3 and wild-type plants were transplanted to a paddy field in Jiangsu Province, China. During the mature stage, yield parameters were scored for 3 (2014), 20 (2015), 15 (2016) and 40 (2017) plants per transgenic line and wild-type rice for statistical analysis. Tiller angle was measured between the main culm and the first side tiller. Tiller angles of the ZmPIF3 and wild-type plants were calculated at 60 days and 120 days. The unit area yields of ZmPIF3 transgenic and WT rice in the field were tested in 2017. The planting density was 30 plants/m2. The unit area yields of ZmPIF3 transgenic and WT rice were calculated for four replicates in different regions and > 100 plants per replicate.
Statistical analyses
Statistical analyses were performed using SPSS version 16.0 software (SPSS, Chicago, IL, USA) and analyzed with a Student’s t test. Significant differences are indicated by asterisks in the figures.
Results
ZmPIF3 reduces stomatal opening and decreases water loss in rice and Arabidopsis
To determine whether ZmPIF3 was involved in regulating water loss, we performed a water loss assay with wild-type and transgenic plants. We hydroponically cultured 35 seedlings of ZmPIF3 transgenic and wild-type rice transplanted into the same transparent pot filled with hydroponic culture solution. After 3 days, the water level of the ZmPIF3 transgenic rice was significantly higher than the wild-type and vector control rice. Meanwhile, there were no significant differences in the most morphological traits of roots of the transgenic rice compared with the wild-type rice (Supplemental Fig. S1). These results demonstrate that ZmPIF3 can significantly prevent water loss in rice seedlings (Fig. 1a). We also constructed transgenic Arabidopsis which overexpressed ZmPIF3 (Supplemental Fig. S2). A water loss assay was also performed with the Arabidopsis. 30 seedlings of Arabidopsis overexpressing ZmPIF3 and wild-type plants were transferred into the same pot respectively, and 300 ml of water was added. After 4 days, the water level of the ZmPIF3 transgenic Arabidopsis was significantly higher than that of the wild-type plants (Fig. 2a, b). These results are consistent with those obtained in rice. Measurements of the water loss rate from the leaves showed that the leaves of the ZmPIF3 transgenic plants lost less water than the leaves of wild-type plants in both rice and Arabidopsis. (Figs. 1b, 2c). We measured the transpiration rate and stomatal conductance of flag leaves in rice. The transgenic rice also exhibited a lower transpiration rate and stomatal conductance than the wild-type rice (Fig. 1c, d; Supplemental Figs. S3, S4). These results indicate that overexpression of ZmPIF3 leads to a significant decrease in the rate of water loss in both rice and Arabidopsis.
Stomatal movements are finely regulated to control water loss through transpiration in response to environmental changes (Murata et al. 2015). Therefore, we measured the stomatal aperture, density and length of stomatal pores in ZmPIF3 transgenic rice and transgenic Arabidopsis (Figs. 1e–h, 2d–g). In wild-type rice, 9.5% of stomata were completely closed, whereas 33.1, 27.4 and 17.8% were completely closed in the OE-3, OE-5, and OE-11 ZmPIF3 transgenic rice lines, respectively. In addition, 47.3% of stomata were completely open in the wild-type rice, compared with 9.4, 21.3, and 25.5% of open stomata in the respective ZmPIF3 transgenic rice lines (Fig. 1f). We compared guard cell density and guard cell length in wild-type and transgenic rice. The ZmPIF3 transgenic rice showed no significant difference in the density and length of stomatal pores relative to wild-type rice (Fig. 1g, h). In the transgenic Arabidopsis, the results were consistent with those in rice. The density and length of the stomata showed no significant alterations in ZmPIF3 transgenic Arabidopsis relative to the WT plants (Fig. 2d, e). More stomata were completely closed and fewer stomata were completely open in the leaves of the ZmPIF3 transgenic lines compared with the WT Arabidopsis (Fig. 2f, g). These results suggest that increased drought tolerance in ZmPIF3 transgenic plants is largely due to reduced stomatal opening and decreased transpiration.
Overexpression of ZmPIF3 increases ABA sensitivity in rice
ABA promotes stomatal closure to avoid water loss under drought stress (Evans et al. 2001; Desikan et al. 2004). Previous studies have shown that the expression of ZmPIF3 was induced by ABA treatment (Gao et al. 2015); we speculated that ZmPIF3 may be involved in ABA expression. In this study, wild-type and ZmPIF3 transgenic rice seeds were germinated in square dishes containing a gradient of ABA concentrations (0, 5 and 10 µM). In the absence of ABA, seed germination of the different genotypes was similar (Fig. 3a, b). When ABA concentrations increased, germination rates of wild-type seeds concomitantly decreased, while the germination rates of the transgenic rice seeds showed a significant decrease (Fig. 3b). These results suggest that the germination process of the transgenic rice seeds is hypersensitive to ABA. To further clarify the effects of ABA, germination assays were performed under mannitol and NaCl treatments. When these seeds were transferred to 1/2 MS medium containing different concentrations of mannitol or NaCl, transgenic plants showed a lower seed germination percentage than that of wild-type plants (Supplemental Fig. S5). Therefore, these results suggest that ZmPIF3 is positively involved in ABA-modulated germination processes.
We also investigated the ABA sensitivity of transgenic rice at the post germination stage. 2-day-old seedlings were transferred to medium containing different concentrations of ABA (0, 5 and 10 µM) and grown for another 7 and 5 days before plant height and root length, respectively, were assessed. In the absence of ABA, seedling growth of the different genotypes was similar (Fig. 3d, f). In the presence of different concentrations of ABA, root and shoot growth of the transgenic rice was more severely inhibited than in the wild-type (Fig. 3c–f). These results suggest that overexpression of ZmPIF3 can increase ABA sensitivity at post germination stages and indicate that ZmPIF3 is a positive regulator of ABA signaling in rice.
We further investigated endogenous ABA levels in wild-type control and transgenic rice. Under normal conditions, the endogenous ABA level was not significantly different between the wild-type and transgenic rice (Fig. 3g). This result suggests that ZmPIF3 is not involved in ABA synthesis but contributes significantly to the ABA signaling pathway.
ZmPIF3 affects differential gene expression in rice
To explore the molecular mechanisms of water-saving and drought tolerance underlying ZmPIF3 transgenic rice, DGE profiling was performed to determine the differential gene expression profiles between wild-type and transgenic lines at the seedling stage (Supplemental Fig. S6). Under normal growth conditions, a total of 35,073 genes were differentially expressed, with 4002 genes exhibiting a 1.5-fold up-regulation and 2988 genes exhibiting a 1.5-fold down-regulation in ZmPIF3 transgenic rice compared with the wild-type. An important number of genes involved in plant hormone pathways and abiotic stress as well as those related to stomata were found to be differentially regulated in the leaves of ZmPIF3-transgenic rice compared with wild-type rice (Table 1; Supplemental Fig. S7).
OsbZIP88 (LOC_Os12g40920), a rice bZIP transcription factor, was predicted to be an ABA responsive element binding factor (Ji et al. 2009). The expression level of OsbZIP88 was elevated to 3.3-fold in ZmPIF3 transgenic rice. OsRAB16C (LOC Os11g26760) has been shown to be a downstream gene in the ABA signaling pathway (Chen et al. 2012). In this study, expression levels of OsRAB16C were increased to 5.5-fold in ZmPIF3 transgenic rice (Table 1). Moreover, expression levels of OsNCED1, OsNCED2, OsNCED3, OsNCED4, OsNCED5, which are the key genes of ABA biosynthesis, were not found to be significantly different in ZmPIF3 transgenic and wild-type tice (Saika et al. 2007; Zhu et al. 2009). Some ABA signaling genes, such as the PYR/PYL/RCAR, PP2Cs, and SnRK2s genes, were also unchanged in ZmPIF3 transgenic rice under normal conditions (data not shown).
Transcript levels of many well-known drought resistance-related genes including OsRAB16D (LOC Os11g26780), OsPR4b (LOC_Os11g37960) and OsPR4c (LOC_Os11g37950) were also elevated to 4.4-, 2.6- and 2.3-fold, respectively, in ZmPIF3 transgenic rice relative to wild-type rice (Chen et al. 2012; Wang et al. 2011). Further scrutiny of the differentially expressed genes identified a set of EXPANSIN family genes associated with stomata aperture, including OsEXPA2 (LOC_Os01g60770), OsEXPA4 (LOC_Os05g39990), OsEXPB2 (LOC_Os10g40710), OsEXPB3 (LOC_Os10g40720), OsEXPB4 (LOC_Os10g40730), OsEXPB7 (LOC_Os03g01270), OsEXPB11 (LOC_Os02g44108), that were up-regulated at least 1.5-fold (Liu et al. 2012). These results suggest that ZmPIF3 elevated the transcript levels of many stress-resistance and stomata-related genes under normal conditions, thus improving the drought tolerance of the transgenic rice by reducing stomatal aperture.
In addition, other plant hormone pathways and abiotic stress genes also showed up-regulation in ZmPIF3 transgenic rice relative to wild-type rice. For example, brassinosteroid insensitive 1 BRI1 (LOC_Os11g47240), which is involved in the brassinosteroid pathway, was up-regulated and had a higher level in ZmPIF3 transgenic rice compared to wild-type rice. Meanwhile, the expression levels of the jacalin-like lectin domain containing protein SalT (LOC_Os01g24710) was up-regulated to 3.6-fold in the transgenic rice compared to wild-type.
The grain yield of the ZmPIF3 transgenic rice
Because grain yield is the ultimate parameter for crops, we examined several agronomic traits of the ZmPIF3 transgenic rice under normal conditions for four cultivating seasons (2014, 2015, 2016 and 2017) (Table 2; Fig. 4; Supplemental Table S1). Fewer plants were scored in 2014, while the 2015, 2016 and 2017 data exhibited greater statistical rigor. The ZmPIF3 transgenic rice exhibited increased tiller and panicle number. Tillering in rice is one of the most important agronomic traits related to grain production (Li et al. 2003). At the mature stage, the average tiller and panicle number of the ZmPIF3 transgenic rice plants grown in the field was significantly higher compared to the wild-type controls in 2014, 2015, 2016 and 2017 (Table 2; Supplemental Table S1). The filling rate of the ZmPIF3 transgenic rice was decreased, but the reduction appeared to be balanced by the increase in the number of tillers and panicles (Table 2; Supplemental Table S1). Moreover, the unit area yield of the ZmPIF3 transgenic rice was measured in 2017. The unit area yield of the ZmPIF3 transgenic rice did not decrease compared to the wild-type rice (Fig. 5). An additional interesting phenotype of the ZmPIF3 transgenic rice is their significantly wider tiller angle versus that of the wild-type rice, which was also confirmed in three independent lines of ZmPIF3 transgenic rice (Fig. 6d–i). Morphological comparison of ZmPIF3 transgenic rice and wild-type rice demonstrate a difference in tiller angle both in pots and paddy fields at 60 and 120 days (Fig. 6a–c). These results clearly indicate that ZmPIF3 is important for grain yield production by increasing the number of panicles.
Discussion
ZmPIF3 is a positive regulator of drought stress tolerance dependent on ABA signaling to regulate stomata aperture
The plant drought response is a complex process regulated by multiple molecular and cellular pathways. The plant stress hormone ABA is a key mediator in drought stress response because it induces stomatal closing to minimize transpirational water loss (Cutler et al. 2010; Kim et al. 2010; Osakabe et al. 2014). Stomatal responses can regulate water loss from plants to efficiently control the water status of plants and play a crucial role in drought stress tolerance (Murata et al. 2015; Schroeder et al. 2001; Hetherington and Woodward 2003; Huang et al. 2009). The ZmPIF3 transgenic plants showed a lower water loss rate than the wild-type plants in both rice and Arabidopsis (Figs. 1a, b, 2a–c), and significantly promoted stomatal closure and reduced stomatal conductance and transpiration rate in both rice and Arabidopsis (Figs. 1c–h, 2). These adaptations help to better avoid dehydration when soil water becomes limiting. The results indicate that ZmPIF3 plays an important role in the regulation of stomatal closure to control of water loss.
Plant response to drought is largely dependent on enhanced ABA biosynthesis and signaling to regulate both stomatal aperture and gene expression (Cutler et al. 2010; Pizzio et al. 2013; Seiler et al. 2014). In our previous study, we showed that expression of ZmPIF3 is rapidly induced in response to ABA treatment, and the ZmPIF3 transgenic rice exhibited a higher survival rate after exposure to drought stress (Gao et al. 2015). In this study, the ZmPIF3 was significantly able to promote stomatal closure (Figs. 1f, 2g). Therefore, we attempted to investigate the functional relationship between ABA and ZmPIF3. ZmPIF3 transgenic rice exhibited diminished germination rates, plant height and root elongation than wild-type controls under ABA treatment (Fig. 3a–f). These results show that ZmPIF3 transgenic rice exhibited hypersensitivity to ABA in both germination and seedling growth. Meanwhile, the endogenous ABA level in the ZmPIF3 transgenic rice showed no significant change under normal conditions (Fig. 3g). These results show that ZmPIF3 may be involved in the ABA signaling pathway but not involved in ABA biosynthesis. Transcriptomic comparisons could facilitate the identification of key genes and regulatory mechanisms. In this study, the expression level of ABA biosynthesis-related genes OsNCED1, OsNCED2, OsNCED3, OsNCED4, and OsNCED5 (Saika et al. 2007; Zhu et al. 2009) were not found to be significantly changes in ZmPIF3 transgenic and wild-type rice (data not shown). These results are consistent with the results for endogenous levels of ABA content (Fig. 3g), which further suggests that ZmPIF3 is not involved in the ABA biosynthesis pathway. The ABA signaling pathway includes components such as cytosolic ABA receptors, PYL/RCARs, PP2Cs, SnRK2s, and basic leucine zipper (bZIP) transcription factors (Cutler et al. 2010; Umezawa et al. 2010; Kim et al. 2014). In our DGE results, expression of most PYL/RCARs, PP2Cs, and SnRK2s genes was not changed in ZmPIF3 transgenic rice under normal conditions (data not shown). OsbZIP88 encodes a rice bZIP transcription factor and can interact with OsbZIP71 by forming heterodimers with OsbZIP71, while OsbZIP71 may play an important role in ABA-mediated drought and salt tolerance in rice (Liu et al. 2014). The expression level of OsbZIP88 was increased in ZmPIF3 transgenic rice relative to wild-type rice. Moreover, the expression levels of RAB16C, a downstream gene in the ABA signaling pathway (Chen et al. 2012), were increased in ZmPIF3 transgenic rice (Table 1). These results suggest that ZmPIF3 participates as a positive regulator of the ABA signaling pathway.
EXPANSINS are cell wall proteins which mediate cell wall loosening and play an important role in regulation of stomatal aperture and stomatal density (Marowa et al. 2016; Zhang et al. 2011; Wei et al. 2011; Lü et al. 2013). In our DGE results, seven EXPANSIN genes were screened, and the expression of these genes was significantly up-regulated in ZmPIF3-transgenic rice relative to wild-type rice. These results are consistent with the lower transpiration phenotypes of ZmPIF3-transgenic lines (Fig. 1f), These results suggest that ZmPIF3 may participate in regulating stomatal aperture. In addition, ZmPIF3 transgenic rice also exhibited activated stress-responsive gene expression even in the absence of stress treatment, including genes such as OsRAB16D, OsPR4b and OsPR4c (Table 1) (Chen et al. 2012; Wang et al. 2011). The DGE analysis indicated that ZmPIF3 promotes the closing of stomata and a decrease in the transpiration rate by participating in the ABA-dependent signaling pathway, which is responsible for enhanced drought tolerance in ZmPIF3 transgenic plants.
ZmPIF3 transgenic rice improves the number of panicles without affecting the grain yield under normal conditions
The constitutive overexpression of stress-related genes often causes abnormal development and thus a loss in productivity (Dubouzet et al. 2003; Nakashima et al. 2007; Yu et al. 2013). Therefore, it is also important to develop transgenic plants that are tolerant to stresses but also maintain high yields under normal conditions. In rice, grain yield increases are caused by an increased number of tillers and panicles without a substantial change in the number of spikelets per panicle (Ekanayake et al. 1989; Ramegowda et al. 2014; Wei et al. 2014). Here, ZmPIF3 did not affect rice grain yields under normal conditions and increased the number of panicles without a substantial change in the number of spikelets per panicle (Figs. 4, 5; Table 2; Supplemental Table S1). An additional phenotype of ZmPIF3-transgenic plants is their tiller angle is significantly wider than that of WT plants (Fig. 6). We speculated that the wider tiller angle of ZmPIF3 transgenic plants was caused by more growth of the tiller compared with the wild-type plants. The yield results in multiple transgenic lines suggest the broader applicability of ZmPIF3 in crop production.
In our previous study, we characterized a constitutively active form of the transcription factor ZmPIF3 and showed a drought stress-resistant phenotype (Gao et al. 2015). In this study, comparison analysis of ZmPIF3 transgenic rice and wild-type plants revealed that ZmPIF3 is a positive regulator of ABA signaling and regulates drought stress resistance by reducing stomatal opening and transpiration rates. ZmPIF3 not only improved drought tolerance, but also had no effect on the grain yield of rice, demonstrating that it is a promising candidate gene in transgenic breeding of drought resistance plants and crop improvement.
References
Bae G, Choi G (2008) Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol 59:281–311
Boccalandro HE, Rugnone ML, Moreno JE, Ploschuk EL, Serna L, Yanovsky MJ, Casal JJ (2009) Phytochrome B enhances photosynthesis at the expense of water-use efficiency in Arabidopsis. Plant Physiol 150:1083–1092
Boursiac Y, Leran S, Corratge-Faillie C, Gojon A, Krouk G, Lacombe B (2013) ABA transport and transporters. Trends Plant Sci 18:325–333
Castillon A, Shen H, Huq E (2007) Phytochrome interacting factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci 12:514–521
Chen H, Chen W, Zhou J, He H, Chen LB, Chen HD, Deng XW (2012) Basic leucine zipper transcription factor OsbZIP16 positively regulates drought resistance in rice. Plant Sci 193:8–17
Chinnusamy V, Schumaker K, Zhu JK (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot 55:225–236
Cordeiro AM, Figueiredo DD, Tepperman J, Borba AR, Lourenco T, Abreu IA, Ouwerkerk PBF, Quail PH, Oliveira MM, Saibo NJM (2016) Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B. Biochim Biophys Acta 1859:393–404
Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid:emergence of a core signaling network. Annu Rev Plant Biol 61:651–679
Desikan R, Cheung MK, Bright J, Henson D, Hancock JT, Neill SJ (2004) ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J Exp Bot 55:205–212
Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L. encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763
Ekanayake IJ, Datta SKD, Steponkus PL (1989) Spikelet sterility and flowering response of rice to water stress at anthesis. Ann Bot 63:257–264
Evans NH, McAinsh MR, Hetherington AM (2001) Calcium oscillations in higher plants. Curr Opin Plant Biol 4:415–420
Gao Y, Jiang W, Dai Y, Xiao N, Zhang CQ, Li H, Lu Y, Wu MQ, Tao XY, Deng DX, Chen JM (2015) A maize phytochrome-interacting factor 3 improves drought and salt stress tolerance in rice. Plant Mol Biol 87:413–428
Gao Y, Wu MQ, Zhang MJ, Jiang W, Ren XY, Liang EX, Zhang DP, Zhang CQ, Xiao N, Li Y, Dai Y, Chen JM (2018) A maize phytochrome-interacting factors protein ZmPIF1 enhances drought tolerance by inducing stomatal closure and improves grain yield in Oryza sativa. Plant Biotechnol J. https://doi.org/10.1111/pbi.12878
Gonzalez CV, Ibarra SE, Piccoli PN, Botto JF, Boccalandro HE (2012) Phytochrome B increases drought tolerance by enhancing ABA sensitivity in Arabidopsis thaliana. Plant Cell Environ 35:1958–1968
Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424:901–908
Huang XY, Chao DY, Gao JP, Zhu MZ, Shi M, Lin HX (2009) A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev 23:1805–1817
Ji Q, Zhang L, Wang Y, Wang J (2009) Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. J Shanghai Univ 13:174–182
Kim T-H, Böhmer M, Hu H, Nishimura N, Schroeder JI (2010) Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling. Annu Rev Plant Biol 61:561–591
Kim H, Lee K, Hwang H, Bhatnagar N, Kim DY, Yoon IS, Byun MO, Kim ST, Jung KH, Kim BG (2014) Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression. J Exp Bot 65:453–464
Kudo M, Kidokoro S, Yoshida T, Mizoi J, Todaka D, Fernie AR, Shinozaki K, Yamaguchi-Shinozaki K (2016) Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants. Plant Biotechnol J 15:458–471
Lee SC, Luan S (2012) ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ 35:53–60
Leivar P, Monte E (2014) PIFs: systems integrators in plant development. Plant Cell 26:56–78
Leivar P, Quail PH (2011) PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16:19–28
Li XY, Qian Q, Fu ZM, Wang YH, Xiong GS, Zeng DL, Wang XQ, Liu XF, Teng S, Hiroshi F, Yuan M, Luo D, Han B, Li J (2003) Control of tillering in rice. Nature 422:618–621
Liu J, Zhang F, Zhou JJ, Chen F, Wang BS, Xie XZ (2012) Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice. Plant Mol Biol 78:289–300
Liu CT, Mao BG, Ou SJ, Wang W, Liu LC, Wu YB, Chu CC, Wang XP (2014) OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice. Plant Mol Biol 84:19–36
Lü PT, Kang M, Jiang XQ, Dai FW, Gao JP, Zhang CQ (2013) RhEXPA 4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis. Planta 237:1547–1559
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324:1064–1068
Marowa P, Ding AM, Kong YZ (2016) Expansins: roles in plant growth and potential applications in crop improvement. Plant Cell Rep 35:949–965
Murata Y, Mori IC, Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism. Annu Rev Plant Biol 66:369–392
Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630
Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, Tran LSP (2014) ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity. New Phytol 202:35–49
Pizzio GA, Rodriguez L, Antoni R, Gonzalez-Guzman M, Yunta C, Merilo E, Kollist H, Albert A, Rodriguez PL (2013) The PYL4 A194T mutant uncovers a key role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA interaction for abscisic acid signaling and plant drought resistance. Plant Physiol 163:441–455
Ramegowda V, Basu S, Krishnan A, Pereira A (2014) Rice GROWTH UNDER DROUGHT KINASE is required for drought tolerance and grain yield under normal and drought stress conditions. Plant Physiol 166:1634–1645
Saika H, Okamoto M, Miyoshi K, Kushiro T, Shinoda S, Jikumaru Y, Fujimoto M, Arikawa T, Takahashi H, Ando M, Arimura S, Miyao A, Hirochika H, Kamiya Y, Tsutsumi N, Nambara E, Nakazono M (2007) Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8′-hydroxylase in rice. Plant Cell Physiol 48:287–298
Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D (2001) Guard cell signal transduction. Annu Rev Plant Physiol Plant Mol Biol 52:627–658
Seiler C, Harshavardhan VT, Reddy PS, Hensel G, Kumlehn J, Eschen-Lippold L, Rajesh K, Korzun V, Wobus U, Lee J, Selvaraj G, Sreenivasulu N (2014) Abscisic acid flux alterations result in differential abscisic acid signaling responses and impact assimilation efficiency in barley under terminal drought stress. Plant Physiol 164:1677–1696
Sirichandra C, Wasilewska A, Vlad F, Valon C, Leung J (2009) The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action. J Exp Bot 60:1439–1463
Staneloni RJ, Rodriguez-Batiller MJ, Casal JJ (2008) Abscisic acid, high-light, and oxidative stress down-regulate a photosynthetic gene via a promoter motif not involved in phytochrome-mediated transcriptional regulation. Mol Plant 1:75–83
Todaka D, Nakashima K, Maruyama K, Kidokoro S, Osakabe Y, Ito Y, Matsukura S, Fujita Y, Yoshiwara K, Ohme-Takagi M, Kojima M, Sakakibara H, Shinozaki K, Yamaguchi-Shinozaki K (2012) Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress. Proc Natl Acad Sci USA 109:15947–15952
Umezawa T, Nakashima K, Miyakawa T, Kuromori T, Tanokura M, Shinozaki K, Yamaguchi-Shinozaki K (2010) Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol 51:1821–1839
Wang FF, Lian HL, Kang CY, Yang HQ (2010) Phytochrome B is involved in mediating red light-induced stomatal opening in Arabidopsis thaliana. Mol Plant 3:246–259
Wang NL, Xiao BZ, Xiong LZ (2011) Identification of a cluster of PR4-like genes involved in stress responses in rice. J Plant Physiol 168:2212–2224
Wei P, Chen S, Zhang X, Zhao P, Xiong Y, Wang W, Wang X (2011) An a-expansin, VfEXPA 1, is involved in regulation of stomatal movement in Vicia faba L. Chin Sci Bull 56:3531–3537
Wei SY, Hu W, Deng XM, Zhang YY, Liu XD, Zhao XD, Luo QC, Jin ZY, Li Y, Zhou SY, Sun T, Wang LZ, Yang GX, He GY (2014) A rice calcium-dependent protein kinase OsCPK9 positively regulates drought stress tolerance and spikelet fertility. BMC Plant Biol 14:133
Yoshida T, Mogami J, Yamaguchi-Shinozaki K (2014) ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Curr Opin Plant Biol 21:133–139
Yu LH, Chen X, Wang Z, Wang SM, Wang YP, Zhu QS, Li SG, Xiang CB (2013) Arabidopsis enhanced drought tolerance1/HOMEODOMAIN GLABROUS11 confers drought tolerance in transgenic rice without yield penalty. Plant Physiol 162:1378–1391
Zhang XQ, Wei PC, Xiong YM, Yang Y, Chen J, Wang XC (2011) Overexpression of the Arabidopsis alpha-expansin gene AtEXPA 1 accelerates stomatal opening by decreasing the volumetric elastic modulus. Plant Cell Rep 30:27–36
Zhang DP, Zhou Y, Yin JF, Yan XJ, Lin S, Xu WF, Baluška F, Wang YP, Xia YJ, Liang GH, Liang JS (2015) Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation. J Exp Bot 66:6371–6384
Zhu GH, Ye NH, Zhang JH (2009) Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis. Plant Cell Physiol 50:644–651
Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 31771686); Natural Science Foundation of the Jiangsu Province (No. BK20161334); State Key Laboratory of Crop Biology (No. 2016KF03). We also thank another financial support for the Priority Academic Program Development of Jiangsu Higher Education Institutions and State Key Laboratory of Crop Biology.
Author information
Authors and Affiliations
Contributions
YG and JC conceived and designed the experiments; YG, MW, MZ, WJ, EL, NX and CZ performed the experiments; YG, DZ and JC analyzed the data and wrote the paper.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Gao, Y., Wu, M., Zhang, M. et al. Roles of a maize phytochrome-interacting factors protein ZmPIF3 in regulation of drought stress responses by controlling stomatal closure in transgenic rice without yield penalty. Plant Mol Biol 97, 311–323 (2018). https://doi.org/10.1007/s11103-018-0739-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-018-0739-4