Ectopic expression of secretory peptide PdEPF3 in Arabidopsis confers drought tolerance with reduced stomatal density

Drought is one of the most prevalent environmental stresses that affect plant growth and development. Improvement in drought tolerance is associated with reduced stomatal density and higher water use efficiency (WUE). In this study, an epidermal patterning factor (EPF), PdEPF3, from a fast-growing poplar clone, NE-19 [Populus nigra × (Populus deltoides × Populus nigra)], was characterized. Quantitative reverse transcription polymerase chain reaction showed that the transcription of PdEPF3 was induced by drought. We further found that the transgenic Arabidopsis overexpressing PdEPF3 had an earlier seedling germination and longer primary roots under osmotic stress treatments, compared with the WT and mutant epf1-1. In addition, ectopic overexpression of PdEPF3 in Arabidopsis plants was able to enhance drought tolerance. This observation was associated with the reduced stomatal density of transgenic plants, which would limit transpiration and reduce water loss, consequently improving the WUE of plants. Interestingly, the reduction of stomatal density in transgenic plants overexpressing PdEPF3 did not affect their photosynthetic capacity. These results indicate that PdEPF3 could be used in transgenic breeding to enhance plant drought tolerance.


Introduction
At present, 65% of the world's agroforestry production is affected by drought, with developing countries accounting for more than 80% of this proportion [1]. For the sustainable development of agriculture and environment, it is important to identify key genes that enhance drought tolerance without reducing yield or biomass [2]. With the development of molecular biology and genetics, more researchers are focusing on the use of technology to improve the water use efficiency (WUE) of plants. Water use efficiency is defined as the ratio of carbon assimilation to transpiration, or the ratio of biomass production to water consumption. Compared with other engineering techniques for water saving, breeding new varieties with a high WUE and drought resistance has several advantages, such as: low investment and sustainable efficiency [3].
As a part of the adaptation towards environmental stresses, plants have evolved delicate regulatory mechanisms to sense and respond to these stresses [4]. The most obvious and important management strategies for plants to cope with drought stress are minimizing water loss through closure of stomata and reduction in stomatal density [2]. Stomata, which are surrounded by two guard cells, are the primary regulators of carbon dioxide uptake and water loss in plants [5]. As most of the water absorbed by a plant is lost to the air through transpiration, the regulation of stomatal behavior (movement and development) is an important factor affecting WUE and drought resistance [6]. The balance between carbon gain through photosynthesis and water loss through transpiration affects WUE. Under drought stress, plants tend to close their stomata to conserve water and improve WUE [7].Water use efficiency is an important basis for the functional identification of novel drought gene resources. Several studies have demonstrated the possibility of improving drought tolerance and WUE by reducing the stomatal density on leaves. By using genetic manipulation or mutation to reduce stomatal density, improvements in WUE have been achieved across several model species, including Arabidopsis [8][9][10][11], poplar [12], wheat [13], barley [14], and tobacco [2].
Over the past decade, a good understanding of the signaling pathways that control the formation of stomata has been developed. A central feature in stomatal development is the "one-cell spacing" rule, in which two stomata are separated by at least one intervening nonstomatal epidermal cell [15]. This spacing is presumed to provide the necessary position for gas exchange and ion exchange [5]. During the process of plant growth and development, the stomata or their precursors ensure "one-cell spacing" pattern by sending a cell-to-cell signal to the undifferentiated neighbor cells, which, in turn, modulates their cell division [16]. Cell-to-cell signaling is essential for many processes in plant growth and development. Secreted peptides are important cell-to-cell signaling components that coordinate and specify cellular functions in plants. Extracellular and cross-tissue communication via known peptide ligands, such as EPF1, EPF2, and STOMAGEN, received by TMM or ERECTA family receptors, could potentially provide the spatial information to position stomata away from the underlying cells [17][18][19].
We had previously reported the isolation of PdEPF1 and PdEPF2 from poplar, and demonstrated their role in improving the WUE and conferring drought tolerance in the plant [12,20]. In this manuscript, an additional member of the EPF family, PdEPF3, was isolated, and its functions were analyzed. We demonstrate that the ectopic overexpression of PdEPF3 reduced the stomatal density and water loss mediated by, thus increasing drought tolerance.

Plants material and growth conditions
Arabidopsis mutant and transgenic lines were used in the Columbia-0 (Col-0) background. Arabidopsis thaliana ecotype Col-0 was used as the wild-type (WT) control. Seeds of Col-0 and the T-DNA insertion mutant epf1-1 (SALK_137549) were obtained from the ABRC. All the seeds were surface sterilized for 1 min in 75% ethanol followed by 10 min in 1% NaClO and five washes in sterilized distilled water. Seeds were sown on half-strength Murashige and Skoog (1/2 MS) [21] plates supplemented with 3% (w/v) sucrose and 0.6% (w/v) agar. The seeds were stratified for 2 days at 4°C before transfer to a growth room maintained at 22°C under a 16 h / 8 h (white light/dark) photoperiod, 80% relative humidity, and 150 μmol m −2 s −1 irradiation. Seven days after the germination, the seedlings were transplanted and grown at a density of four plants per pot (7 × 7 × 6.5 cm) containing a mixture of rich soil and vermiculite (2:1, v/v), under a 16/8 light/dark photoperiod at 22°C, 70% relative humidity, and 150 μmol m −2 s −1 irradiation.

PdEPF3 gene cloning, transformation, and expression analysis
Total RNA isolation was performed using the CTAB reagent method [22]. First-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase and an oligo (dT) primer (Promega, Madison, WI, USA) in accordance with the manufacturer's instructions. The full-length cDNA of PdEPF3 was retrieved by homologous cloning, using gene-specific primers FW 5'-ATGAGAAAAACCAAAGCCATGAAG-3' and RV 5'-TCATGGGACAGGGTAAGACTT-3'. The PCR products were cloned into the pMD18-T cloning vector (Promega, Madison, WI, USA) and were subsequently subjected to sequencing analysis. The amplified PdEPF3 open reading frame was inserted into the XbaI and BamHI sites in the pBI121 vector under the control of the cauliflower mosaic virus (CaMV) 35S promoter, to generate the fusion construct 35S:PdEPF3.
The constructs were introduced into Agrobacterium tumefaciens (strain GV3101) and transferred into Arabidopsis plants (wild type and mutant) using the floral dip method [23]. The transformed lines were screened on hygromycin-supplemented media (containing 50 mg/L hygromycin) to generate independent transgenic lines. The homozygous T3 lines were used for further analysis.
For subcellular localization of PdEPF3 in leaf epidermal cells, PdEPF3-GFP fusion proteins were observed using a confocal laser scanning microscope (DMI6000 CS; Leica, Wetzlar, Germany).

RT-PCR and qRT-PCR analysis
Total RNA from each sample was extracted by the CTAB method. Quantitative real time-PCR (qPCR) was performed as described by previously [24]. SYBR GREEN Master Mix (TianGen Bio Inc., Beijing, China) was used to monitor the kinetics of PCR product formation during qPCR. UBQ was used as an internal control to quantify the relative transcript level of PdEPF3. Gene-specific primers were designed using the Primer6 (PrimerE, Ivybridge, UK), and the primer sequences are shown in Tab. S1.

Morphological characterization
To compare the morphological differences among oxPdEPF3, WT, epf1-1, and epf1-1/ oxPdEPF3 plants under well-watered condition, rosette leaves were removed from 22-day-old seedlings, and the leaf area was computed using PHOTOSHOP CS5. The plant height was measured at 25 and 40 days after germination.

Characterization of germination rate and root length
For osmotic treatment experiments, Arabidopsis seeds were sown on 1/2 MS agar plates supplemented with 200 mM mannitol and 25 mM PEG6000 [25]. Three independent batches of seeds were used to compare the germination rate among oxPdEPF3, WT, epf1-1, and epf1-1/oxPdEPF3 plants. For the root length assay, 5-day-old seedlings grown on 1/2 MS agar plates were transferred to vertically oriented 1/2 MS agar plates supplemented with 200 mM mannitol and 25 mM PEG6000 and grown for 10 days before the root length was measured. Six seeds from each line were used to compare the root lengths. All experiments were repeated three times.

Drought stress and water loss measurements
For testing the phenotype under drought stress, seedlings grown on 1/2 MS medium for 7 days were transferred to a mixed soil and grown for 3 weeks with sufficient watering. Each was planted with four seedlings planted. The plants were then subjected to drought stress treatment by withholding irrigation for 12 days. The plants were rewatered for 1 week after significant differences in wilting were observed. All the pots were subjected to under identical conditions within a growth chamber. The photographs were taken after withholding water or rewatering for the indicated times. To evaluate the water loss rate of the plants, rosette leaves of transgenic plants, WT, epf1-1, and epf1-1/oxPdEPF3 growing under normal conditions for 3 weeks were detached. The leaves were harvested from the same stage of different plants (about 0.5 g) and weighed immediately on a piece of weighing paper. They were then placed on a laboratory bench and weighed every 30 min, with three replicates. The percentage of fresh weight lost was calculated based on the initial weight of the leaves.

Measurement of leaf stomatal density
To measure the stomatal density, leaves of the same age and from the same elative position were sampled from the overexpression transgenic plants, WT, epf1-1, and epf1-1/oxPdEPF3 grown under identical conditions. The fixed sample method was used as described previously [20]. The fixed samples were dried and then set up on an aluminum plate for coating with gold. These samples were observed by scanning electron microscopy (Hitachi S-3400 N; Chiyoda-ku, Tokyo, Japan). For statistical analysis of stomatal density, three leaves were sampled for each plant, and three individual plants were included in each group. Different letters indicate significant differences at α = 0.05 (one-way ANOVA). Data are presented as mean ±SE.

Leaf gas-exchange measurements
The net photosynthetic (A) and transpiration rates (E) were measured using the Li-6400 Portable Photosynthesis System (Li-Cor, Lincoln, NE, USA). Data were recorded during 9 a.m. -11 a.m. At the time of data recording, the ambient CO 2 concentration was 400 μmol mol −1 , the photosynthetic photon flux density was 600 μmol m −2 s −1 , and the chamber temperature was 24°C. Water use efficiency (WUE) was calculated from instantaneous values of A and E.

Molecular characterization of PdEPF3
We identified a putative poplar peptide which was homologous to PdEPF1 [12], and designated this peptide as PdEPF3 (GenBank accession number MF461306). A comparison showed that PdEPF3 shared a high sequence similarity and clustered with PdEPF1, and similar to PdEPF1 and AtEPF1, it also contains eight conserved cysteine residues at the C-terminal (Fig. 1A). The PdEPF3 cDNA is 387 bp in length and encodes 128 amino acid residues with a predicted molecular mass of 14.25 kDa and an isoelectric point of 9.53 (http://web.expasy.org/compute_pi/). The subcellular localization results showed that the localization of PdEPF3-GFP could be in cytosol, plasma membrane, or cell wall. An empty vector with GFP served as the positive control (Fig. 1B).

Expression profile of PdEPF3
The transcription of PdEPF3 was tissue-specific. Transcripts were detected in roots, stems, young leaves, mature leaves, and senescent leaves of NE-19 under nonstress growth conditions. The results showed that PdEPF3 was expressed more highly in young leaves and mature leaves than in senescent leaves, but was the lowest in root ( Fig. 2A). Under drought stress, PdEPF3 transcription initially increased but subsequently decreased during days 3-12 of drought treatment (Fig. 2B).

Analysis of transgenic Arabidopsis overexpressing PdEPF3
To elucidate the in vivo functions of PdEPF3, the 35S:PdEPF3 construct was transformed into WT Arabidopsis and the epf1-1 mutant, to generate overexpression and complementation lines, respectively. We raised nine independent transgenic lines, of which three were selected, based on the qRT-PCR results. As expected, PdEPF3 transcripts were undetectable in the WT plants. Three of the overexpression lines (Lines 2, 4, and 5) showed higher transcript levels than the other overexpression lines (Fig. S1), and were designated as oxPdEPF3#1, #2, and #3.

Morphological analysis of oxPdEPF3 lines under well-watered condition
At 22 days after germination, the oxPdEPF3 lines had larger leaves than WT and epf1-1. The rosette leaf area of the oxPdEPF3 lines was 13.7-21.4% and 44.16-53.93% larger than that of WT and epf1-1, respectively (Fig. 3A). As shown in Fig. 3B, after 25 days after germination, the inflorescence length of oxPdEPF3 lines was longer than that of WT and epf1-1. Forty days after germination, the inflorescence length of all plants reached 400 mm. This result indicates that the growth rates of oxPdEPF3 lines, WT, and epf1-1 were different, but they eventually became consistent in morphology.

Overexpression of PdEPF3 enhanced the tolerance of Arabidopsis to drought stress
To study the drought-tolerance mechanism, various experimental setups were developed. The seeds of transgenic lines, WT, epf1-1, and the complemented line epf1-1/oxPdEPF3 were sown on 1/2 MS culture with 200 mM mannitol and 25 mM PEG6000, for osmotic stress. After 7 days, in the presence of 200 mM mannitol, the oxPdEPF3 lines had more vigorous germination (74.7-81.9%) than WT (63.3%) and the mutant (42.2%) (Fig. 4A).
In the presence of 25 mM PEG6000, oxPdEPF3 lines had more vigorous germination (83.4-84.8%) than WT (38.1%) and the mutant (28.7%) (Fig. 4B). However, in comparison, all the seeds of transgenic lines, WT, epf1-1, and epf1-1/oxPdEPF3 had sprouted after 7 days under normal conditions (data not shown). In addition, when 5-day-old seedlings grown on 1/2 MS medium were transferred to vertical agar plates containing 1/2 MS medium supplemented with 200 mM mannitol and 25 mM PEG6000, the primary root lengths of the 10-day-old seedlings were also different. In the presence of 200 mM mannitol, oxPdEPF3 lines had considerably longer (1.6-and 1.8-times, respectively) primary roots than that of WT and the mutant, epf1-1 (Fig. 4C). In the presence of 25 mM PEG6000, oxPdEPF3 lines had longer (1.5-and 1.7-times, respectively) primary roots than that of WT and the mutant epf1-1 (Fig. 4D).
The assays of water loss from detached leaves showed that the epf1-1 plants lost water much more quickly than the WT plants, and that the oxPdEPF3 plants lost water much more slowly than both WT and epf1-1 plants (Fig. 5A). The fresh weight of transgenic plants was reduced to 73.6% for Line 1, 68.5% for Line 2, 66.9% for Line 3, 59.9% in wild-type plants, and 45.6% in the epf1-1 mutant.
The capacity of oxPdEPF3 lines to respond to drought stress was investigated next. As shown in Fig. 5B, the oxPdEPF3 plants did not show any difference in their phenotype compared with the WT and mutant plants under the control conditions, but they did show much stronger tolerance to drought stress than WT and the mutant plants under the drought stress. These results confirmed that PdEPF3 overexpression can greatly improve the drought resistance of transgenic Arabidopsis.

Ectopic overexpression of PdEPF3 decreases stomatal density and improves WUE in Arabidopsis
To clarify the physiological mechanism responsible for the increased drought tolerance of PdEPF3-overexpression lines, the stomatal density on the abaxial leaf epidermis of 3-week-old rosette leaves was studied. The stomatal density in the oxPdEPF3 plants was 20-24% less than that of the WT plants, and almost 50% less than that of the epf1-1 mutant plants (Fig. 6A). The reduced stomatal density apparently contributes to the reduced rate of water loss in transgenic plants. The epf1-1 mutant also showed a defect in the one-cell-spacing rule of stomatal pattern, marked by the red arrow in Fig. 6B. This overexpression phenotype of PdEPF3 indicated that this protein is capable of negatively affecting stomatal patterning. Reduced stomatal density is known to affect water and CO 2 exchange [26]. Therefore, we measured the photosynthetic and transpiration rates of all lines and found that the oxPdEPF3 plants maintained a significantly higher photosynthetic rate and lower transpiration rate than the WT and epf1-1 plants under drought stress (Fig. 7A,B). Consequently, the WUE is higher for the transgenic plants (Fig. 7D). These findings indicated that the lower stomatal density was one of the reasons for the higher drought tolerance of 35S:PdEPF3 Arabidopsis compared with the WT and epf1-1 mutant.

Discussion
Water is essential for plant growth because it plays an important role in many cellular functions. Biomass production and accumulation are negatively affected by water deficit because such conditions reduce the leaf cell turgor, restrict cell expansion, and postpone development [22]. Researchers have found that most of the water loss from leaves occurs through the stomata, which is known as stomatal transpiration, and reduced transpiration is an important aspect of plant drought resistance [27]. Regulation of stomatal behavior has been an active area of research on drought stress. Stomatal density may be a target trait for plant engineering to improve the photosynthetic capacity, particularly in future high-CO 2 atmospheres [9,26,28,29].
In this present study, PdEPF3 was cloned from poplar. According to the analysis, PdEPF3 shared high amino acid sequence similarity with and clustered with PdEPF1, which improves WUE and confers drought tolerance in transgenic poplar by decreasing   the stomatal density [12]. PdEPF3 also contains eight conserved cysteine residues at the C-terminal, similar to PdEPF1 and AtEPF1 (Fig. 1A).
Under abiotic stress conditions, the expression of many genes is induced, and their products have important roles in stress responses and tolerance. In the current study, expression analysis showed that PdEPF3 was concomitantly induced in response to water deficit in the NE-19 seedlings (Fig. 2B). Overexpression of PdEPF3 in Arabidopsis exhibited earlier seedling germination time and longer primary roots under the osmotic stress conditions (Fig. 4). Roots are the organs that absorb water and nutrients from soil. Therefore, the root growth state directly effects the growth and development of the whole plant [30]. In addition, PdEPF3 overexpression in transgenic plants also maintained a higher leaf water potential than that of wild type and epf1-1 mutant under water-deficit conditions (Fig. 5). Stomata are the main portals of gas exchange between the plant above-ground organs and the atmosphere and have a strong effect on the characteristics associated with photosynthesis and transpiration [31]. AtEPF1 is a well-characterized negative regulator of stomatal development in Arabidopsis thaliana [32]. Through ectopic overexpression of PdEPF3, we created Arabidopsis transformants with reduced stomatal density: the oxPdEPF3 plants had 20-24% lower stomatal density than the WT plants, and almost 50% lower density than the epf1-1 mutant (Fig. 6A). Decreased stomatal density was a primary factor underlying the enhanced drought resistance in oxPdEPF3 Arabidopsis. This decrease in stomatal density and conductance most likely allows plants to fine tune their responses to the environment and exercise a better control of CO 2 assimilation and water utilization [33,34]. Consistent with this, transgenic Arabidopsis overexpressing PdEPF3 showed significantly higher WUE under drought conditions (Fig. 7D). Theoretically, stomatal density is negatively correlated with both photosynthesis and transpiration, whereas, our results indicated that the oxPdEPF3 plants maintained a higher photosynthetic rate and lower transpiration rate than those of the WT and epf1-1 plants under drought stress (Fig. 7A,B). Moreover, Franks et al. [10] reported that, in Arabidopsis, a reduction in stomatal conductance via reduced stomatal density in EPIDERMAL PATTERNING FACTOR 2 (EPF2)-overexpressing plants increased both instantaneous and long-term WUE, without significantly altering the photosynthetic capacity [10]. In rice, it has been reported that an Arabidopsis home domain-leucine zipper transcription factor, Enhanced Drought Tolerance / HOMEODOMAIN GLABROUS11 (EDT1/HDG11), was able to confer drought tolerance and increase the grain yield in transgenic plants. Furthermore, the improved drought tolerance was associated with a more extensive root system, reduced stomatal density, and higher water use efficiency [35]. The Arabidopsis GT-2 LIKE 1 loss-of-function mutations (gtl1) result in increased water deficit tolerance and higher integrated WUE by reducing the daytime transpiration without a reduction in biomass accumulation [9]. Therefore, the above-reported traits exemplify the adaptive mechanisms in plants that help them survive under drought stress without loss of productivity or yield.
In this study, we identified and isolated PdEPF3, and showed that its expression in Arabidopsis plants improved drought tolerance. Our findings suggest that this gene may be useful in the breeding of drought-tolerant plants, and in protecting and optimizing the plant yields under future drier environments. Further experiments are needed to generate additional transgenic poplars, and to elucidate the regulatory mechanisms of drought tolerance in woody plants.