Overexpression of an aquaporin gene PvPIP2;9 increased biomass yield and protein content, improved drought tolerance and water use eciency and affected other PIP2 genes’ expression in switchgrass (Panicum virgatum L.)

Background: Switchgrass ( Panicum virgatum L.) is a prime candidate for non-grain-based bioenergy feedstock production. Improved drought tolerance and higher water use eciency are important for its successful eld establishment and production, especially on marginal lands. Aquaporins are key channels and regulators for water transportation and maintenance of cellular water status. In this study, the functional role of an aquaporin gene, PvPIP2;9 , in switchgrass was studied. Results: Expression of PvPIP2;9 was regulated by diurnal oscillation and osmotic stress. Constitutive over-expressing PvPIP2;9 in switchgrass signicantly improved its leaf length, plant height, above-ground biomass, biomass protein contents, and cellulose contents in stressed plants. Under 21 days of drought treatment, transgenic plants showed less electrolyte leakage rates, but higher relative water contents, photochemical eciencies, and chlorophyll contents, indicating that PvPIP2;9 positively regulated plant drought tolerance and water use eciency. Moreover, expression patterns of all 14 switchgrass PIP2 subfamily genes were checked during the drought treatment, and the result showed that over-expressing PvPIP2;9 also affected transcript levels of most other PIP2 genes. Conclusions: Together, this study showed that improved biomass yield, drought tolerance and higher water use eciency can be achieved by manipulating the expression level of PvPIP2;9 and also suggested PIP2 subfamily genes were transcriptionally regulated in a coordinated manner.


Introduction
Switchgrass (Panicum virgatum L.) is a warm season tall perennial grass originated in North America and has been used as a pasture forage (1) and a non-grain-based bioenergy feedstock (2). A previous study showed that all tested switchgrass ecotypes suffered severe biomass reduction (75-80%) with water stress at -4 MPa (3). To minimize competition with primary food crop production for land use, much of switchgrass production are on less productive marginal lands where irrigation is often limited or unavailable during prolonged drought periods. Therefore, improved drought tolerance and higher water use e ciency (WUE) are important targeting traits for switchgrass molecular breeding.
Aquaporin family genes play important regulatory roles in water movement through the symplastic pathway and maintenance of cellular water homeostasis in plants (4). Based on their sequence compositions, aquaporins can be divided into ve types, including tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), X-intrinsic proteins (XIPs), and plasma membrane intrinsic proteins (PIPs), among which PIPs might be the main gateways controlling water permeability (5). Furthermore, PIPs can be categorized into two phylogenetic subgroups, PIP1 and PIP2, according to their main structural differences. A number of PIP2s were found possessing high water channel activities and played indispensible roles in water transport (6,7). Several individual studies have reported that over-expressing PIP2 subfamily genes improved plant drought tolerance, including FaPIP2;1 in tall fescue (Festuca arundinacea) (8), PIP2;5 in Populus (Populus tremula × Populus alba) (9), TaAQP7 in wheat (Triticum aestivum) (10), and HvPIP2;1 in barley (Hordeum vulgare) (11). These results suggested that some PIP2 genes functioned as positive regulators in plant drought tolerance. Yet, in some other cases, over-expressing PIP2 genes compromised drought tolerance. For examples, over-overexpression of a soybean (Glycine soja) stress-inducible PIP2 gene (GsPIP2;1) in transgenic Arabidopsis (Arabidopsis thaliana) increased plant sensitivity to dehydration (12). Overexpression of PIP1;4 and PIP2;5 in both Arabidopsis and tobacco (Nicotiana tabacum) resulted in rapid water loss under dehydration stress as well as retarded seed germination and seedling growth under drought stress (13). Lee et al. (14) reported that activating an E3 ligase led to degradation of PIP2;1 and improved drought tolerance in Arabidopsis. In short, some PIP2s were functional in water transport, yet their roles in plant drought tolerance are diversi ed, and how these PIP2 family genes interacted and cooperated in maintaining plant water status was still unclear.
There are 68 aquaporin genes in switchgrass, including 7 PIP1 and 14 PIP2 subfamily members (15), and none of their functions has been characterized so far. In our previous study, over-expressing an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (LOV1) in switchgrass resulted in smaller leaf angles, improved drought tolerance and higher WUE. Comparative microarray analysis revealed that there were 105 signi cantly differentially-expressed genes, among which PvPIP2;9 (microarray probe ID: KanlowCTG00810_at; Phytozyme accession No.: Pavir.Ba02478) was the only differentially-expressed and up-regulated aquaporin gene in the transgenic plants (16,17). Therefore, we hypothesized that PvPIP2;9 plays an important role in the regulation of switchgrass drought tolerance. In the current work, the expression pattern and functional role of PvPIP2;9 in switchgrass drought tolerance were studied, and effects of drought and over-expression of PvPIP2;9 on other PIP2 family genes were also measured.

Results
Phylogenetic analysis and expression pattern of PvPIP2;9 According to the switchgrass genomic sequence information (Panicum virgatum v4.1, DOE-JGI, http://phytozome.jgi.doe.gov/), we cloned PvPIP2;9 from switchgrass. PvPIP2;9 had 286 amino acids with a predicted molecular mass of 29.87 kD. As a typical aquaporin protein, PvPIP2;9 had six conserved transmembrane (TM) domains and two nucleosome assembly protein (NAP) domains (Fig.1a). A phylogenetic tree comprising of PvPIP2;9 and its orthologs in rice (Oryza sativa), maize (Zea mays), and Arabidopsis showed that PvPIP2;9 was most closely related to OsPIP2;4 with 97% of amino acid similarity (Fig. 1b). At the organ/tissue level, the transcript level of PvPIP2;9 in roots was the highest, which was ~6-10 times higher than those in leaves and leaf sheaths, and was ~10-20 times higher than those in in orescence of rachis, orets, vascular bundle, and internodes (Fig. 2a). In leaves, the expression of PvPIP2;9 showed a clear diurnal change that increased after dawn, reached to its maximum level in the middle of daytime and declined thereafter to its basal level at the end of the daytime (Fig. 2b). Furthermore, we measured its relative expression level in plants under 20% PEG6000 (water potential: -0.735 MPa) and 100 μM ABA treatments compared with the control sampled at the same time point. Treating switchgrass plants with PEG6000-induced osmotic stress resulted in signi cantly increased expression of the gene within 2 hr, and such an activated expression was transient that returned to its initial levels after 4 hrs of treatment and remained at its basal level thereafter (Fig. 2c). However, ABA treatment did not activate but suppressed the expression of PvPIP2;9 (Fig. 2d), indicating that the osmotic stress-induced expression of PvPIP2;9 was likely independent of the ABA signal.
Over-expressing PvPIP2;9 led to improved drought tolerance, water use e ciency, higher biomass yield and crude protein contents in switchgrass The PvPIP2;9 was over-expressed in switchgrass under driven of the maize ubiquitin promoter by Agrobacterium-mediated genetic transformation. A total of 11 putative transgenic lines were generated. The presence of T-DNA in these putative lines was con rmed by PCR amplifying a fragment of HPTII gene (conferring hygromycin-resistance) (Fig. 3a). Three representative transgenic lines (line-1, -7 and -9) with over-expressed PvPIP2;9 and positive GUS staining signals (Fig. 3b&C) were chosen for further phenotypic analyses.
Transgenic lines and tissue culture-regenerated WT plants of the same age were vegetatively propagated by splitting and growing single tillers under the optimum growth condition. After two and a half months of growth, single tillers of WT and the transgenic lines proliferated into ve to seven tillers, and all PvPIP2;9-OX lines showed signi cantly longer leaf length, taller plant height, and higher above-ground biomass yield (fresh weight and dry weight) than those of WT (Table 1).
These switchgrass plants were treated by withdrawing water to evaluate whether over-expressing PvPIP2;9 affected drought tolerance. After 28 days of drought period, WT became severely wilted that even the newly emerged leaves at the top withered and turned yellow, while plants of all transgenic lines still remained 1-2 green leaves at the top in each tiller. After re-watering, WT plants did not recover but completely died off, while all PvPIP2;9-OX lines recovered back with new green expanding leaves (Fig.  5a). During the drought treatment, the pot soil water content dropped from -0.48 MPa to -0.83~-0.85 Mpa, -1.35~-1.20 MPa, and ~-1.65 MPa after 7, 14, 21 and 28 days of water withdrawal, respectively, while those under well-watered condition had a relative constant soil water content of -0.48-0.5 MPa (Fig. 5b). Five physiological parameters, including photochemical e ciency (Fv/Fm), chlorophyll (Chl) content, electrolyte leakage (EL), leaf relative water content (RWC), and WUE were measured in WT and transgenic lines during the drought treatment. As shown in gure 5c-g, there was no signi cant difference among these physiological parameters between WT and PvPIP2;9-OX lines when under the well-watered condition. Yet, under the drought treatment, signi cant differences were observed after 21 days of treatment for all ve physiological parameters that transgenic plants had signi cantly lower EL, but higher RWC, Fv/Fm, Chl contents and WUE than those of the WT (Fig. 5c-g). WUE of plants after 28 days of water holding was not measured because leaves of WT were completely wilted already. Furthermore, values of WUE, EL and RWC of the transgenic lines were also related to their relative expression of PvPIP2;9. For example, transgenic line-7 had the highest expression level of PvPIP2;9 in leaves, while line-1 had the least. After 28 days of drought treatment, line-7 showed the lowest EL and the highest RWC, while the opposite was true for line-1. These results supported that over-expressing PvPIP2;9 signi cantly improved switchgrass drought tolerance and WUE associated with higher photochemical e ciency, higher membrane stability, higher Chl content, and better leaf water status under this prolonged stress.
To understand whether over-expressing PvPIP2;9 also affected biomass feedstock quality, we measured total sugar, cellulose, hemicellulose, lignin and crude protein contents in above-ground biomass of WT and transgenic lines. As shown in table 2, all transgenic lines had signi cantly higher crude protein content than WT no matter the plants were grown under the optimum soil water or after 21 days of drought. It is also notable that the tested plants showed varied levels of total sugar, cellulose, hemicelluloses and lignin under the optimum soil water condition; yet after 21 days of drought, transgenic lines had signi cantly higher cellulose.
Over-expression of PvPIP2;9 affected expression patterns of other PIP2 genes during drought treatment To understand whether over-expressing PvPIP2;9 also affected other PIP2 subfamily genes, we further measured relative expression levels of all PvPIP2 genes during the water-withdrawal treatment in WT and the three transgenic plants.
Firstly, it was notable that expression of PvPIP2;9 per se was responsive to the long-term drought stress but was distinctively different in leaves and roots: the relative expression of PvPIP2;9 increased ~3 times in WT leaves, but reduced to 20% in WT roots during the drought treatment. While in transgenic lines, relative expression levels of PvPIP2;9 were ≥200 times higher than those of WT in leaves, and ≥2 times higher than those of WT in roots (the only exception was that in line-1 and -7 before drought treatment where they were at similar expression levels to that in WT). Yet, during the drought treatment, expression levels of PvPIP2;9 decreased in leaves but gradually increased 6-13 folds in roots in the two transgenic lines (Fig. 6).
Secondly, as shown in WT, most PIP2 subfamily genes were responsive to the drought treatment. For examples, transcriptional levels of PvPIP2;3, PvPIP2;4, and PvPIP2;5 increased in response to decreasing soil water content in both leaves and roots; while those of PvPIP2;11 and PvPIP2;13 showed increased expression pattern only in roots but not in leaves (Fig. 7).

Discussion
There are 68 aquaporin genes in switchgrass (15). Yet none of these genes was functionally characterized so far. Based on our previous study, we predicted that PvPIP2;9 might be an important aquaporin contributing to the leaf water status in switchgrass. Current results in this study indicate that PvPIP2;9 positively regulated switchgrass drought tolerance and WUE.
PvPIP2;9 is an important aquaporin regulating plant water status in switchgrass Under drought treatment, transgenic switchgrass demonstrated signi cantly improved drought tolerance and WUE under this prolonged drought stress. The association between aquaporin genes' expression and WUE has been documented before. For examples, over-expressing a stress-inducible aquaporin gene (NaAQP1) in tobacco, tomato (Lycopersicon esculentum) and Arabidopsis all increased their WUE and photosynthesis under both optimal and salt stress conditions (18). And over-expressing a TIP-type aquaporin (AQUA1) in white poplar (Populus alba) also improved the plant's WUE and RWC (19). In this study, we found transgenic plants had signi cantly higher WUE associated with lower EL, higher Fv/Fm, Chl content, and RWC than WT after prolonged drought treatment (Fig. 5). Lower EL value indicated better cell membrane integrity, higher Fv/Fm and Chl content further corroborated that leaves of transgenic plants were bringing their functions into better play, and the higher RWC in transgenic plants con rmed that PvPIP2;9 positively contributed to cellular water status of leaves when the soil water content was low. We reasoned that the higher WUE in transgenic plants should be another important reason for the improved drought tolerance that saved water loss from evapotranspiration, which in turn contributed to better cellular water status as re ected in leaf RWC and the maintenance of integral cell membrane system and photosynthesis system. Expression level of PvPIP2;9 affected plant growth and biomass feedstock quality in switchgrass Under the well-watered condition, constitutive over-expression of PvPIP2;9 in switchgrass did not signi cantly alter the WUE and leaf relative water content though, suggesting that over-expressing of this aquaporin gene did not signi cantly affect water transportation or water status when there was su cient soil water supply. Yet, transgenic lines did show signi cantly longer leaf length, taller plant height and higher above-ground biomass than those of WT. Another interesting nding with the transgenic switchgrass was their higher protein contents no matter they were grown under the optimum or drought condition, and showed signi cantly higher cellulose content after 21 days of drought. The higher biomass protein content was not essential for lignocellulosic biofuel feedstock but was a highly desirable trait when switchgrass was used as forage feedstock. To our knowledge, such an effect on biomass protein and cellulose contents was not well emphasized or recorded for a PIP gene before.
It was reported that leaf elongation rate responded to rapid changes in evaporation and soil water availability in an even considerably quicker manner than transpiration and leaf water potential (e.g. 30 min vs. 1-2 h), and small ux of water potentials could cause rapid decline of simulated leaf elongation rate (20). Nada & Abogadallah (21) reported that reduced WUE and decreased RWC at mid-day in rice even under well-watered paddy eld which was associated with inadequate expression of aquaporin genes, and such an suppression could be eliminated upon removal of radial barriers to water ow in roots. We reasoned that these WT and transgenic plants be challenged by temporary water de cit (e.g. at noon time) even though they were regularly watered and grown under optimum condition in greenhouse. While due to the effect of PvPIP2;9 over-expression, transgenic plants were less challenged with temporary uxes of unfavorable leaf water potential. Decades earlier, effect of water stress on protein contents in maize cultivars with contrasting drought tolerance has been reported that the drought tolerant cultivar showed higher protein contents than the susceptible one (22). Effects of short-term drought (4 or 7 days of drought) in switchgrass (23) and long-term drought (28 days) in miscanthus (24) were reported that their cell wall components were affected by drought stress to various degrees. For example, cellulose content was signi cantly lower in drought treated miscanthus (24). The relatively higher cellulose content in stressed PvPIP2;9 transgenic switchgrass was at least partially explained by the better plant water status during the drought treatment. PvPIP2;9 and other PvPIP2 subfamily genes were ne-tuned and interacted at the transcriptional level OsPIP2;4 was the closest rice orthologous gene of PvPIP2;9. Although it is still unclear whether OsPIP2;4 plays a regulatory role in plant drought tolerance or not, it was reported that the expression of OsPIP2;4 showed a clear diurnal expression pattern and OsPIP2;4 had high water channel activity (6). PvPIP2;9 also had a similar expression pattern to OsPIP2;4 that its diurnal transcription level was in conjunction with the activity of plant diurnal water transport. The diurnal changes of aquaporin genes have also been reported for a few other PIP2 genes in other plant species, such as HvPIP2;1 in barley (25), ZmPIP2;1 and ZmPIP2;5 in maize (25), and OsPIP2;4 and OsPIP2;5 in rice (6). Such an expression pattern suggested that water uptake in leaves during the daytime demand higher transcriptional level of this aquaporin gene. Furthermore, the expression of PvPIP2;9 in leaves was transiently up-regulated by osmotic stress, but down-regulated by exogenous applied ABA treatment in leaves. Dehydration caused di culty in root water absorption with lower osmotic potential in the rst place, and caused less water evapotranspiration rate by inducing stomata closure and induced ABA production at a later stage (26). The transiently increased expression of PvPIP2;9 upon PEG-treatment could be a direct response to the water-de cit due to PEG-induced shortage of root water absorption. And the ABA-induced suppression of PvPIP2;9 in leaves could be due to the quick response of stomata closure that in turn helped balancing leaf water status. In short, the expression pattern of PvPIP2;9 was highly responsive to plant water status, which was in congruent with the gene's function in the regulation of plant water status, indicating that regulation of the PIP2 gene at the transcription level was important for switchgrass.
Upon the recognition of PIPs' contributions to water transport and cellular water homeostasis, there is an interest to understand the full picture of how these PIPs interacted and coordinated with each other in these cellular processes. From the perspective of the protein-protein interactome, previous studies on two maize PIP1 and three PIP2 subfamily genes showed that ZmPIP1;1 and ZmPIP1;2 could form heterodimers but showed no activity of osmotic water permeability (27); yet this ZmPIP1-ZmPIP2 interaction was required for PIP1 tra cking to plasma membrane (28). At the same time, such physical interaction between PIP1-PIP2 (e.g. ZmPIP1;2-ZmPIP2;1) was required for their functions to form consolidated water channels (27). A more recent work studying on interactomes of PIP1;2 and PIP2;1 proteins, using the approach of immunoprecipitation and quanti cation by mass spectrometry (IP-MS), revealed that these two proteins shared about 400 interacting proteins (29). This big interacting protein mass likely behaved as a "platform" for recruitment of various proteins likely involved in transport activities including those responding to osmotic and oxidative treatments (29). At the post-translational level, 12 out of 13 Arabidopsis PIPs were found to have varied types of post-translational modi cations including phosphorylation, methylation, deamidation, and acetylation in response to environmental stress (30). Meanwhile, at the post-transcriptional level, it was reported that microRNAs (miRNAs) were endogenous modulators of multiple aquaporin genes in human (31). Another study in Arabidopsis also reported that salinity treatment invoked a simultaneous transcriptional repression and protein internalization of PIP2;7 (32). Yet, it was less recognized that, at transcriptional level, expression of these PIP2 genes were also inter-affected. There are 14 PvPIP2 genes in switchgrass in reference to the current switchgrass genome database ("Panicum virgatum v4.1, DOE-JGI, http://phytozome.jgi.doe.gov/"). In this study, we found that over-expressing PvPIP2;9 in switchgrass signi cantly affected expression of many other PIP2 genes (Fig. 7). We reasoned these changes of other PIP2 genes at the transcriptional level might be due to feedback effect of cellular status in the transgenic plants because of potential functional redundancy between these PIP2 genes, or due to post-transcriptional regulation of PIP2 genes (e.g. targeted by miRNA on certain common sequence among these PIP2 genes), which was yet to be studied in the future. Overall, our current results indicate that there was a complicated interacting network of PIP2s at the transcriptional level as well. Together with previous ndings, PIPs likely responded to environmental constraints at multiple levels of gene regulation to adjust plant water status.
As mentioned earlier, PvPIP2;9 was the only aquaporin gene with signi cantly increased expression in the LOV1 transgenic plants that showed improved drought tolerance (16,17). We also tested whether or not the LOV1 transcription factor could directly bind to the -2 Kb promoter region of PvPIP2;9 using yeast onehybrid test. However, our results showed that there was no transactivation effect of LOV1 on the PvPIP2;9 promoter (data not shown), suggesting that LOV1 indirectly activated the expression of PvPIP2;9 in switchgrass.

Conclusion
A PIP2 gene was cloned and functionally characterized for the rst time in switchgrass in this study. PvPIP2;9 positively contributed to plant water status, enhanced plant protein contents of above-ground biomass, and signi cantly improved switchgrass cellulose contents, drought tolerance, and WUE in stressed plants. Moreover, the result together with previous reports supported that PIP2s were coordinately regulated at multiple levels, including transcriptional, post-transcriptional and posttranslational levels, to adjust plant water status. Results of this study highlight the importance of the aquaporin gene, PvPIP2;9, and the complexity of PIP2 family genes in the regulation of switchgrass water status. Such information will be useful for switchgrass molecular breeding toward improved drought tolerance and higher WUE.

Plant materials and growth condition
Seeds of an elite switchgrass line, HR8, originally selected from the lowland ecotype 'Alamo' was used in this study (33). In the qPCR experiment to study PvPIP2;9 expression pattern, 4-wk-old plants were cultured in 1/2 Hoagland solution and grown in a growth chamber with a 12-hour (hr) light/dark cycle and accurately controlled temperature [30/25°C (day/night)] and light intensity (photosynthetically active radiation at 750 µmol·photons m -2 · s -1 ). In other experiments, switchgrass plants were grown in grown in clay loam soil mixed with sand (1:1) in 1.1×10 -2 m 3 pots in the greenhouse at Nanjing Agricultural University (Nanjing, China) with temperatures set at 30/20 ± 3 °C (day/night) and the photoperiod set at 14/10 hr (day/night).

qRT-PCR analysis
The second fully expanded leaves from the top were sampled for relative gene expression level and physiological parameter analyses. To detect the diurnal oscillation of PvPIP2;9, the rst sampling time was set at the dawn for consecutive 40 hr with four hr internals in-between. For stress treatments, plants were grown in 1/2 Hoagland solution containing 20% polyethylene glycol (PEG) 6000 (Huada, Shantou, China) and 100 μM ABA according to Yuan et al. (34), and sampled after 0, 0.5, 1, 2, 4, 8 and 12 hr after the treatment.
The total RNA was isolated using OMEGA E.Z.N.A. ® plant RNA Kit. The rst strand cDNA was synthesized with 1 μg RNA using the PrimeScript TM RT reagent Kit (Takara, Dalian, China) with the Perfect Real Time gDNA Eraser (TaKaRa). The qRT-PCR was performed using SYBR Green Master Mixes on a Roche LightCycler ® 480 II machine. The qRT-PCR was performed with three biological replicates and two technical replicates, and the qPCR program set as follows: 10 min at 95 °C for initial denaturation, and 40 cycles (95 °C for 15s, 58 °C for 15 s, and 72 °C for 20 s). Relative expression levels of PvPIP2;9 were calculated using the 2 -ΔΔCT method with PvACTIN as the reference gene (35). Primers used in this study were shown in Additional le 1: Table S1.

Gene cloning and vector construction
According to the switchgrass genomic sequence information (Panicum virgatum v4.1, DOE-JGI, http://phytozome.jgi.doe.gov), we cloned the gene from gDNA for its functional characterization. In brief, the gene was ampli ed from switchgrass genomic DNA using PCR, cloned into the vector pENTR/D and sequenced. Then we sub-cloned the gene into the Gateway-compatible binary vector pVT1629 (35) using LR reaction (Invitrogen). The resultant vector, pVT1629-PvPIP2;9, harboring the PvPIP2;9 driven under maize ubiquitin promoter and the UidA (GUS) reporter gene under CaMV 35S promoter, was transformed into the Agrobacterium tumefaciens strain 'AGL1' through electroporation.

Switchgrass genetic transformation
Switchgrass line 'HR8' was used for Agrobacterium-mediated genetic transformation and the transformation procedure was the same as reported before (33). Hygromycin B (Sigma) at 50 mg/L was used to select against the non-transformed calli. Regenerated plants from independent calli were regarded as putative transgenic lines which were further veri ed by GUS staining and PCR for the detection of HPT gene present in the T-DNA.

Drought treatment of WT and transgenic plants
Two independent transgenic lines and tissue culture-regenerated wild-type (WT) plants were propagated by splitting single tillers grown at the optimum condition. Plants grown from a single tiller for two and a half months reached E4 stage (36) and were used for drought treatment by withdrawing water. After 28d of drought, the treated plants were re-watered to observe their re-growth status. At the same time period, normally-watered plants were used as controls. A soil water content detector (Mini Trase Kit 6050X3; Soil Moisture Equipment Corp., Santa Barbara, CA) was used to monitor the soil water content (SWC) in 0-8 cm deep soil layer of each pot. And soil water potential was determined using ERS-water potential and temperature meter (Yibaiyi Mechanical and Electrical Equipment Co., Ltd., Wenzhou, China). The correlation between soil water content and soil water potential was shown in Additional le 2: Figure S1.
Biomass feedstock quality analysis After 21 d of treatment, the above ground of WT and transgenic plants were collected and dried in a 70℃ oven and then ground for feedstock quality analysis. The amount of total sugar was determined by the phenol sulfuric acid reagent method (37) with slight modi cations. In brief, 0.05 g samples were added to the mixture of 10 ml ddH 2 O and 3 ml HCl and incubated in water bath set at 100℃ for 1 h. Then 0.5 ml supernatant was transferred to a 10 ml tube and mixed with 0.5 ml ddH 2 O, 1 ml 5% phenol, and 5 ml sulfuric acid. The reaction mixture was incubated at 30℃ for 20 min in a water bath and then the absorbance was quanti ed at 490 nm. The quantity of total sugar was based on a standard curve generated with known sugar concentrations.
Cellulose content was estimated using the anthrone method (38). Brie y, 0.05 g samples were mixed with 35 ml 60% H 2 SO 4 in a 50 ml tube and incubated at ice bath for 30 min. After ten times dilution, the absorbance of supernatant was quanti ed at 620 nm. Microcrystalline cellulose (Avicel) was used as a standard for the standard curve generation.
Hemicellulose and lignin content were measured by hydrochloric acid hydrolysis method and sulfuric acid method, respectively (39). For hemicellulose analysis, 0.1 g sample was mixed with 10 ml 80% calcium nitrate and boiled on a heater for 5 min. The mixture was then centrifuged and the supernatant was discarded. After rinsed three times with ddH 2 O, 10 ml 2 M HCl was added to the mixture and boiled for another 45 min. The supernatant was neutralized using NaOH and mixed with DNS reagent. The mixture was incubated in water bath at 100℃ for 5 min and then absorbance of supernatant was measured at 520 nm. For lignin, 0.1 g sample was washed using 10 ml 1% acetic acid and the mixture of ethanol and ether (1:1) and then was dried in a water bath set at 100℃. The sample was incubated with 72% H 2 SO 4 for at least for 16 h to remove cellulose. The precipitate was then mixture with 10 ml 10% H 2 SO 4 and 0.1 M potassium dichromate and incubated in water bath set at 100℃ for 15 min. The lignin content was then quanti ed after mixed with 5 ml 20% KI and 1 ml 0.5% starch solution by titration using 0.2 M sodium thiosulfate.
The Kjeldahl procedure was used to determine the total nitrogen (TN) content, and the crude protein content was calculated by multiplying TN by 6.25 (40).

Measurement of physiological parameters
Leaf membrane stability was evaluated by measuring the electrolyte leakage (EL) (41) according to a method described before (42). In brief, leaves were excised and cut into 3 cm segments. Then the leaves were incubated in 35 ml distilled deionized water. Centrifuge tubes were shaken on a shaker for 24 hr at room temperature, and the initial level of EL (C i ) was measured using a conductance meter (Thermo Scienti c, Beverly, USA).Then the leaf tissue was killed by autoclaving at 121°C for 15 min, and then incubated for 24 h on a shaker for measuring the maximum conductance (C max ) of the solution. Relative EL was calculated as EL = (C i /C max ) × 100%.
The leaf relative water content (RWC) was determined according to the method described by Hu et al. (43) with modi cations. In brief, RWC was determined using fresh fully expanded leaves (~0.2 g). Leaf samples were detached from the plants and immediately weighed to determine the fresh weight (FW). Samples were placed into covered centrifuge tubes lled with water for leaves to reach full hydration. After approximately 24 h at 4 °C, leaf samples were blotted dry with paper towels and weighed to determine the saturated weight (SW). Leaf tissue was then dried in an oven at 65 °C for 72 hr to determine dry weight (DW). Leaf RWC was calculated as RWC = (FW -DW) / (SW -DW) × 100.
The ratio of the variable fluorescence (Fv) to the maximal fluorescence (Fm) (Fv/Fm) was used to represent leaf photochemical e ciency (Oxborough and Baker, 1997). The Fv/Fm ratio was determined using a fluorescence meter (Dynamax, Houston, TX, USA) as described before (42). And chlorophyll content were measured using the DMSO extraction method as described before (42).
Leaf instantaneous WUE was calculated by measuring leaf net photosynthetic rate (Pn) and transpiration rate (Tr) using the LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE). The area of leaves enclosed in the leaf chamber was determined on a scanner, which was then used to calculate the Pn and Tr values. The WUE was calculated as Pn/Tr.

Statistical analysis
Data in this study were statistically analyzed using one-way ANOVA, and their means were compared by Duncan test at the signi cance level of 0.05 by using SPSS20.0. Tables   Table 1. Over-expression of PvPIP2;9 affected above-ground growth of WT and transgenic plants. Three independent transgenic lines and tissue culture-regenerated wild-type (WT) plants were propagated by splitting single tillers grown at the optimum condition for two and a half months reached E4 stage. Then the tiller number, leaf width, leaf length, and plant height were measured. The above ground fresh and dry weight were tested from those plants after another 28 days growth. Data are means ± SE (n=8), and different letters represent significant difference at P <0.05.       Relative expression of other PIP2 subfamily genes in leaves and roots during 21 days of drought using qRT-PCR. Data of qRT-PCR were converted into the heatmap using R-package software (version 3.3.1) and the quantitative color scheme was based on log2 of each PIP2 subfamily genes' relative expression levels.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx