Overexpression of OsPIL1 enhanced biomass yield and saccharification efficiency in switchgrass

Plant Science 276 (2018) 143–151

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Overexpression of OsPIL1 enhanced biomass yield and saccharification efficiency in switchgrass ⁎⁎

Jianping Yana,1, Yanrong Liua,1, Kexin Wanga, Dayong Lic, Qingquan Hud, , Wanjun Zhanga,b,

T ⁎

a

Department of Grassland Science, China Agricultural University, Beijing, 100193, PR China National Energy R&D Center for Biomass (NECB), China Agricultural University, Beijing, 100193, PR China c State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, PR China d Yunnan Animal Science and Veterinary Institute, Kunming, 650224, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: OsPIL1 Switchgrass Biomass Saccharification efficiency

Switchgrass (Panicum virgatum L.) is a herbaceous cellulosic biofuel plant with broad adaptability. However, the intrinsic recalcitrance of biomass and limited land for switchgrass planting hinder its utilization as feedstock for biofuel ethanol production. The OsPIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 1) gene encodes a basic helix-loop-helix transcription factor. Its expression is induced by light, which facilitated the expression of cell wall-related genes, promoted cell elongation and resulted in longer internode in rice. Here, we introduced the OsPIL1 gene into switchgrass by Agrobacterium-mediated transformation with the aim of improving biomass yield of transgenic switchgrass plants. The transgenic plants were verified by PCR, Southern-blotting, RT-PCR and qRT-PCR tests, respectively. The transgenic plants overexpression of OsPIL1 showed increased plant height and biomass yield. Microscopy analysis showed that the length of epidermal cells of transgenic plants was longer than that of wild type. OsPIL1 overexpressed transgenic switchgrass plants also released more soluble sugar after enzymatic hydrolysis, indicating improved saccharification efficiency. The results suggest OsPIL1 can be used as a useful molecular tool in improving plant biomass and saccharification efficiency with the purpose of plant fiber biofuel ethanol production.

1. Introduction Energy requirement is increasing dramatically with the development of modern society. A large amount of fossil fuel was consumed all over the world each year. Such as only in China, coal consumption was increased by more than 2.3 billion tons over the past 10 years, and oil consumption was grown by 4.3% at the end of 2016 [1]. However, fossil energy is non-renewable and emits noxious gas and carbon dioxide during burning. The emission of carbon dioxide was reported to be concerned with global warming [2]. Besides the adverse effects of consuming fossil fuel, countries in the world are also facing with emerging energy crisis and environmental deterioration. The development and/ or utilization of renewable energy resource is thought to be an alternative of fossil energy, which is environment friendly. Herbaceous biomass plants are rich in cellulose and have the characteristics of growing fast, short life cycle and wide adaptability,

and they are the most potential renewable clean energy resources [3,4]. Planting herbaceous energy plants also can maintain and restore biodiversity [5]. Switchgrass (Panicum virgatum L.) is a kind of poaceae C4 perennial herbaceous plants. It has been regarded as the herbaceous energy model plant for its ecological adaptability, broad resistance, rapid growth, and high biomass yield (about 20 t/hm2) [6–9]. In order to maximize the potential of utilizing switchgrass as a biomass feedstock, the bioenergy feedstock development program of U.S. Department of Energy (DOE) had decided to concentrate research resources on switchgrass since 1991 [4]. Highly efficient genetic transformation system of switchgrass had been established and further optimized [[10,11,12]10,11,12]. To promote the production of plant fiber fuel ethanol, characteristics of switchgrass had been successfully modified by genetic transformation by enhancing biomass yield [13–15], and/or reducing lignin content to improve the sugar release [16–18]. The phytochrome interacting factors (PIFs), belonging to basic

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Corresponding author at: Department of Grassland Science, China Agricultural University, Beijing, 100193, PR China. Corresponding author. E-mail addresses: [email protected] (J. Yan), [email protected] (Y. Liu), [email protected] (K. Wang), [email protected] (D. Li), [email protected] (Q. Hu), [email protected] (W. Zhang). 1 These authors contributed equally to this work. ⁎⁎

https://doi.org/10.1016/j.plantsci.2018.08.012 Received 24 April 2018; Received in revised form 14 August 2018; Accepted 23 August 2018 Available online 26 August 2018 0168-9452/ © 2018 Published by Elsevier B.V.

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Fig. 1. Molecular identification of OsPIL1 transgenic (TG) switchgrass plants. (A) Schematic-map of T-DNA region of plasmid pZH01-OsPIL1-OX that used to Agrobacterium-mediated genetic transformation; LB: left border, RB: right border, 35S: CaMV35S promoter, nos: nos terminator, the black bar indicates the region of hpt gene used as a probe for Southern-blotting. (B) PCR tests of the OsPIL1 transgenic plants; the expected size of the DNA fragment (206 bp) was shown; M: marker; P: positive control, DNA of plasmid pZH01-OsPIL1-OX was used as a template, WT: negative control, the DNA of the WT plant was used as a template. (C) Southern-blotting tests of the integration of exogenous OsPIL1 gene. (D) RT-PCR analysis of the expression of the OsPIL1 gene in the TG lines; the switchgrass internal Ubiquitin gene (Ubq) was used as a template loading control. (E) Quantitative real-time PCR tests of the relative expression of OsPIL1 gene in TG plants; error bars represent stand error (SE) of the means (n = 3).

MEGA5.1. Amino acid sequences of switchgrass PvPIL1 and rice OsPIL1 were aligned by DNAMAN6.0. The full-length (1233 bp) of rice OsPIL1 cDNA sequence was amplified by reverse transcript PCR (RT-PCR) using a pair of primers (OsPIL1_OX_XbaF: 5′-TCTAGAATGGCGATTTGCAGCACGGA -3′ and OsPIL1_OX_SalR: 5′-GTCGACCTAAATTCCATCAGAGGTTG -3′), and then cloned into the Xba I and Sal I site of the binary vector pZH01, driven by the CaMV35S promoter [31]. The T-DNA region of the expression vector also contained a hygromycin phosphotransferase gene (hpt) as a selectable marker gene under the control of the CaMV35S promoter. The binary vector was transferred into A. tumefaciens stain EHA105 for Agrobacterium-mediated genetic transformation [12].

helix-loop-helix (bHLH) sub-family, were identified in Arabidopsis thaliana firstly [19]. PIFs were regarded as systems integrators in plant development [20], participating in multiple signaling pathways such as promoting hypocotyls elongation [21,22] and shade-avoidance response in the phytochrome-mediated light-signaling pathway [23,24]. PIFs also reportedly inhibited seed germination [25], promoted stem elongation at elevated temperature in hormonal signaling pathways [26], and regulated the circadian system by mediating metabolic signals [27]. Six PIF-like genes were found in rice [28]. The OsPIL1 was also involved in cell elongation by activating downstream cell wallrelated genes in rice [29]. Ectopic expression of OsPIL1 in Arabidopsis thaliana reportedly enhanced hypocotyl elongation [30]. Based on the previous reports, we assumed that OsPIL1 might be used to improve switchgrass biomass yield for its growth-promoting function. To assess the hypothesis, we introduced the OsPIL1 into switchgrass plants. The results showed that overexpression of OsPIL1 in switchgrass increased biomass yield and promoted saccharification efficiency of transgenic plants compared to non-transgenic control plants.

2.2. Plant materials The resistant and non-transgenic control switchgrass (c.v Alamo) plantlets regenerated from the same callus line derived from a single mature seed, were obtained by following the method reported by Liu et al. [12]. The transgenic plants were cultured in a greenhouse with natural light and temperature ranging from 25 °C to 35 °C at night and day.

2. Materials and methods 2.1. Cloning of the OsPIL1 gene and construction of the plant expression vector

2.3. Identification of the transgenic plants

According to the cDNA sequence (LOC_Os03g56950) of rice OsPIL1 gene, other PIFs (PvPIL1, AtPIF3, ZmPIF4, SiPIF4, BsPIF5 and PhPIF5) derived from different plant species (Panicum virgatum, Arabidopsis thaliana, Zea mays, Setaria italica, Brachypodium sylvaticum and Panicum hallii) were obtained in the website http://phytozome.jgi.doe.gov/ by performing a BLASTN search. These PIFs amino acid sequences were used to construct a Neighbor-Joining phylogenetic tree by using

Plant genomic DNA was extracted from leaves with CTAB method [32]. PCR tests were conducted with a pair of primers specific to OsPIL1 (PIL-F: 5′-GAAGATTCAGACAGTCGCAGTG-3′; PIL-R: 5′-ATGCCTTGTC GGTCTTGTTG-3′) with a standard PCR program at an annealing temperature of 55 °C. The PCR positive transgenic plants were also subjected to Southern-blotting tests according to Liu et al. [12]. Twenty five μg genomic DNA was digested with restriction enzyme BamH I. 144

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Fig. 2. Phenotypic analyses of OsPIL1 transgenic switchgrass plants. Phenotype of six-month-old WT and TG plants in greenhouse. (B) Comparison of plant height of six-year-old WT and TG plants in greenhouse. (C) Comparison of inflorescence length and corresponding internode length in WT and TG plants of heading stage; the scale bars stand for 10 cm. (D) Average length of internode of six-year-old WT and TG plants in greenhouse. (E) Longitudinal sections of the first internode of WT and TG plant; the labeled bracket represents one epidermal cell; the scale bars stand for 100 μm. (F) Comparison of the length of epidermal cells of the first internode in WT and TG plants. Error bars represent SE of means (n at least = 3); different letters indicate significant differences between WT and TG plants (P < 0.05).

(PIL-F and PIL-R) were specific to the OsPIL1 gene. The annealing temperature for RT-PCR was 55 °C, and 30 reaction cycles were given. QRT-PCR reactions were performed using the SYBR green supermix (Takara RR420) and the EcoTM Real-Time PCR System (Illumina, EC100-1001). The 2−ΔΔCT method [33] was used to determine the relative expression levels of OsPIL1 gene. A ubiquitin gene (AP13CTG25905) of switchgrass was used as the internal control for RNA normalization [34].

Table 1 Phenotypic analyses of mature WT and TG plants in greenhouse. Plants

Inflorescence length (cm)

Stems diameter (mm)

Tiller number

Internode number

WT TG9 TG13 TG12 TG11 TG29

64.06 65.51 78.51 69.43 73.89 68.01

2.79 3.00 3.34 2.98 3.26 3.05

4.13 8.25 8.25 5.00 7.50 4.50

3-5 4-5 3-6 4-6 4-5 4-5

± ± ± ± ± ±

2.47 0.83 0.33 2.02 0.51 1.64

e de a c b cd

± ± ± ± ± ±

0.03 0.09 0.16 0.27 0.12 0.07

c bc a bc ab ab

± ± ± ± ± ±

0.95 3.69 2.75 2.71 1.73 2.38

2.4. Phenotypic analysis of transgenic plants Note: each value is mean ± SE (n = 4); different letters indicate significant difference between WT and TG plants at P < 0.05 by AVOVA analysis.

WT and TG plants proliferated from tillers were all fully developed after growing in flowerpots (Φ = 25 cm, h = 25 cm) for six months in a greenhouse. We measured morphological characters of mature tillers (R3 stage, inflorescence fully emerged and peduncle completely elongated before flowering) [35], including plant height, internode and inflorescence length, internode number, tiller number, and stem diameter (middle of the second node, from the ground up). Mature tillers were harvested for fresh weight and dry weight measured. Each transgenic line and wild type plant utilized for measurement included four biological replicates. Each replicate included at least four mature tillers of a plant.

Probe labeling and hybridization procedure was performed following the manufacturer’s instructions in DIG High primer DNA labeling and detection starter kit II (Roche Applied Science. Cat. No. 11585614910). To analyze the expression of OsPIL1 gene, we extracted the total RNA of the transgenic (TG) and wild type (WT) switchgrass plants with a plant RNA extraction kit (Takara Co. Dalian, China). One microgram of total RNA was treated with DNase I and used for cDNA synthesis following the protocol of a reagent kit (Takara RR047 A). The cDNA was used as a template for RT-PCR and QRT-PCR analyses. Primers 145

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Fig. 3. Biomass yield of OsPIL1 transgenic switchgrass plants. Comparison of average fresh weight of a single tiller of six-month-old WT and TG plant in greenhouse. (B) Comparison of average dry weight of a single stem (DMS) of six-month-old WT and TG plant in greenhouse. Error bars represent SE of means (n = 4); different letters indicate significant differences between WT and TG plants (P < 0.05).

determined as the ratio of the summation of glucose and xylose released by enzymatic hydrolysis to the amount of sugar (glucose, xylose) present in the cell wall composition. At least three replicates of each sample were measured for each parameter.

Table 2 Measurement of forage quality in mature WT and TG plants stems. Samples

NDF(%)

WT TG9 TG11 TG12 TG13 TG29

66.73 59.87 58.34 60.88 57.62 60.64

± ± ± ± ± ±

ADF(%) 2.94 1.48 2.12 1.90 1.55 1.33

a b b b b b

40.80 36.60 36.20 37.73 35.46 36.22

± ± ± ± ± ±

ADL(%) 1.24 0.67 0.73 0.77 0.60 0.96

a bc c b c bc

5.39 3.86 4.80 5.31 4.29 5.17

± ± ± ± ± ±

0.38 0.45 0.46 0.25 0.52 0.50

a c ab a bc a

2.7. The measurement of forage quality According to the principle of Van Soest herbage analysis using filter bag technology (ANKOM A2000i semi-automatic fiber analyzer), the content of NDF (neutral detergent fiber), ADF (acid detergent fiber) and ADL (acid detergent lignin) in DMS were measured [40]. Five replicates of each sample were given in the experiment.

Note: Each value is mean ± SE (n = 5). Different letters of the same column indicate existing significant differences between WT and TG plants (P < 0.05).

2.5. Microscopy 2.8. QRT-PCR tests of the relative expression of cell elongation related genes Following the description of Sun et al. [36], the first (top) internode of transgenic plants and wild type plants at heading stage (R1 stage, inflorescence begins to emerge before spikelet fully emerged in reproductive stage) [35] were used to make paraffin sections. Two internodes of each plant were sampled for making paraffin sections. Pictures of transverse and longitudinal sections were taken and three sections from one tiller were obtained. The length of the epidermal cells of WT and TG plants was measured in the pictures of longitudinal sections using Image-Pro Plus6.0. Two sections from different sample of each plant were treated as one replicate. Three replicates were given in the experiment for statistical analysis.

It was reported that expression of Expansin S1 (Os03g0336400), Extensin protein-like (Os03g0637600), 1-aminocyclopropane-1-carboxylate (1-ACC) oxidase (Os09g0451400) and Homeobox Protein 2 (At4g16780) were possibly regulated by OsPIL1 in Oryza sativa and Arabidopsis thaliana [29,30]. The homologous of these genes in switchgrass was sought out in the website http://phytozome.jgi.doe. gov/ by performing a BLASTN search. The corresponding gene ID was Pavir.J13116.1, Pavir.Ib04078.1, Pavir.J25986.1 and Pavir.Gb01134.1, respectively. Total RNA was extracted from the first node (from the top, three tillers) of WT and TG plants in boot stage. After reverse transcription, the first strand cDNA was used for QRT-PCR analysis. The primers used were listed in Table S1. Three biological replicates were given.

2.6. Carbohydrate composition and saccharification efficiency measurement

2.9. Statistical analysis

The tillers of R3 stage were harvested, and after removing inflorescence, leaves and sheaths, the stems were then dried at 65℃ for 48 h. The dried stems were ground and went through 1 mm sieve as the dry materials of stems (DMS). The carbohydrate composition of DMS was analyzed according to a two-stage acid hydrolysis [37]. Three hundred mg DMS were soaked with 3 ml 72% sulfuric acid and incubated in 30°C for one hour, then diluted with ultrapure water from 72% to 4% of the sulfuric acid concentration, and then incubated at 121°C for one hour. The supernatant was used to test the carbohydrate composition by analyzing the released monomeric sugar content. For enzymatic hydrolysis efficiency determination, the DMS was digested by direct exposure to enzyme mixtures of cellulose and cellobiase (Imperial Jade Biotechnology Co., Ltd) for 72 h (as unpretreated samples) or pretreated with 0.25 M NaOH at 50°C for 2 h, and then after washing with water and 0.2 M sodium acetate buffer solution (as pretreated samples) the samples were exposed to the same enzyme mixture for 72 h in a 50°C, 80 rpm shaking incubator. The supernatant was used to test the monomeric sugars content [38]. The monomeric sugars (glucose, xylose) were determined by HPLC system equipped with a HiPlex Ca column (7.7 × 300 mm, Agilent Technology, USA), LC-20AT pump (Shimadzu, Japan) and RID-10 A refractive index detector (Shimadzu, Japan) [39]. The enzymatic hydrolysis efficiency (%) was

All the assays were given at least three biological replicates. The data were subjected to one-way analysis of variance (ANOVA) to compare the differences between transgenic plants and wild type plants at P < 0.05 level. The software used for ANOVA was R (www.rproject.org). 3. Results 3.1. Production and verification of the OsPIL1 transgenic switchgrass plants The Neighbor-Joining phylogenetic tree of PIFs derived from different plant species showed highly similarity, which suggested that the OsPIL1 gene was conserved evolutionarily (Fig. S1A). The amino acid sequences alignment between rice OsPIL1 and switchgrass PvPIL1 showed a similarity of 60.7% (Fig. S1B). The results suggested that the function of PIL1 may be conserved between different species. To improve biomass yield of switchgrass, we introduced the OsPIL1 gene into switchgrass (Fig. 1A) by Agrobacterium-mediated transformation [12]. The putative transgenic plants were screened by PCR tests with a pair of primers specific to OsPIL1. A target band (206 bp) was 146

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Fig. 4. Comparison of saccharification efficiency of switchgrass WT and TG plants. (A) Carbohydrate composition of switchgrass WT and TG plants of R3 stage degraded by sulfuric acid. (B) Soluble sugar (glucose and xylose) yield of mature, dry stems of WT and TG plants after directly enzymatic hydrolysis. (C) Saccharification hydrolysis efficiency of WT and TG plants of R3 stage without alkaline pretreatment. (D) Soluble sugar (glucose and xylose) yield from mature dry stems of WT and TG plants after alkaline pretreatment and then enzymatic hydrolysis. (E) Saccharification hydrolysis efficiency of WT and TG plants of R3 stage after alkaline pretreatment. (F) The total sugar yield of each R3 stage tiller released by directly enzymatic hydrolysis and alkaline pretreatment following enzymatic hydrolysis in WT and TG plants. Error bars represent SE of means (n = 3); Difference capital letters and lower case letters indicate existing significant differences between WT and TG plants (P < 0.05); asterisks indicate a statistically significant difference in total soluble sugar yield between WT and TG plants (P < 0.05 or 0.01).

transgenic lines, and the expression level was progressively increased in the order of TG9, TG13, TG12, TG11 and TG29.

shown in the PCR positive transgenic (TG) plant (Fig. 1B). Five positive lines (TG9, TG11, TG12, TG13 and TG29) were chosen for Southernblot tests. As shown in Fig. 1C, all the five PCR positive plants also gave hybridization signals with a fragment of hpt gene as a probe, and no signal was detected in the wild type (WT) plant. Transgenic lines TG9 and TG11 presented only one hybridization band indicated one copy of the insertion of transgene in the genome of the transgenic plant. The other three transgenic lines had multiple copies of insertion of the transgene. Expression level of OsPIL1 gene in the transgenic lines was verified by the RT-PCR and QRT-PCR (Fig. 1D and E). The results demonstrated that the OsPIL1 gene was successfully expressed in the

3.2. Overexpression of OsPIL1 increased plant height and biomass yield of switchgrass TG plants The OsPIL1 TG and WT plants were grown in a greenhouse under the same culture conditions. After growing for six months, TG plants were much taller than WT plants (Fig. 2A). Statistical analysis of plant height showed that the height of TG plants (144.77–161.72 cm) was significantly taller than WT plant (130.85 cm) except for TG9 (Fig. 2B). 147

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Fig. 5. Relative expression tests of OsPIL1 downstream candidate genes. The representative patterns of the relative expression level of 1-aminocyclopropane-1-carboxylate (1-ACC) oxidase (Pavir.J25986.1) (A), Expansin S1 (Pavir.J13116.1) (B), Extensin protein-like (Pavir.Ib04078.1) (C) and Homeobox Protein 2 (Pavir.Gb01134.1) (D). Error bars represent stand error (SE) of the means (n = 3).

the improvement of biomass yield of switchgrass transgenic plants.

Detailed phenotype comparison of the WT and TG plants showed that the increased plant height of OsPIL1 TG plants owing to their longer inflorescences and internodes than that of WT plants (Fig. 2C and D, Table 1), but not the number of internodes (Table 1). The inflorescence length of TG plants (except for TG9) was 68.01–78.51 cm, about 4–14 cm longer than that of WT plant (64.06 cm) and the average internode length of TG plants (16.46-18.63 cm) was remarkably longer than that of WT plant (15.64 cm). We also measured the length of stem epidermal cells of the first internode from top of the switchgrass WT and TG R1 stage plants. As shown in Fig. 2E and F, the epidermal cells of TG plants (about 250 μm) were significantly longer than that of WT plant (200 μm or so). The results suggested that overexpression of exogenous OsPIL1 in switchgrass promoted the elongation of stem epidermal cells and caused a significant increase of plant height. The biomass yield is a very important indicator for switchgrass as a bioenergy plant. In this study, we measured the fresh weight of each mature tiller (R3 stage) and the dry weight of each stem after getting rid of leaves, leaf sheath and inflorescence. As shown in Fig. 3A, the average fresh weight of a single tiller of TG plants (around 12 g/tiller) was significantly heavier than that of WT plant (approximately 8 g/ tiller) except for TG9. And the dry weight of stems (DMS) in TG plants was significantly higher than that of WT plant except for TG9 as well (Fig. 3B). TG plants had thicker stem than WT plants, even some of them had no significant difference with WT plants (Table 1). Overall, the results indicated overexpression of OsPIL1 had a correlation with

3.3. Overexpression of OsPIL1 in switchgrass plants improved forage quality We speculated that the elongated epidermal cells of stems could affect the cell wall composition and forage quality of switchgrass. Hence, we measured the percent of NDF (neutral detergent fiber), ADF (acid detergent fiber) and ADL (acid detergent lignin) of TG and WT plants. The results showed that NDF and ADF content of TG plants was significantly lower than that of WT plant (Table 2). NDF content of TG plants (57.62%–60.88%) showed 8.8%–13.7% reduction compared with that of WT plant (66.73%). ADF content of TG plants was under 38%, which had a 7.5%–13.1% decrease than that of WT plant (40.8%). TG plants also showed lower content of ADL than WT plant, but only the ADL content of TG9 and TG13 was significantly lower than that of WT (Table 2). The results suggested that overexpression of OsPIL1 may have a positive effect on forage quality of the transgenic switchgrass plants. 3.4. Overexpression of OsPIL1 improved saccharification efficiency of switchgrass transgenic plants To test whether overexpression of OsPIL1 affected the saccharification efficiency of transgenic switchgrass plant, the DMS was treated with enzymes mixture of cellulase and cellobiase directly or after 148

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Overexpression of PIF4 and PIF5 promoted hypocotyls elongation of Arabidopsis thaliana seedings, increased biomass yield by delaying flowering and promoting cell expansion [46]. PIF4 has a specific role in improving plant growth and development in high temperature [47]. Overexpressing OsPIL1 in rice increased the elongation of stem cells, enhanced plant height and improved drought resistance [29]. The results we obtained in the experiments proved that the OsPIL1 gene could be used as a molecular tool for switchgrass plant height modification hence to improve biomass yield of TG plants. The cell wall of herbaceous energy plants is the main feedstock of bioethanol production. Therefore, the structure of the cell wall plays an important role in bioethanol production for herbaceous energy plants. The plant cell wall is mostly composed of cellulose, hemicellulose and lignin. However, the complicated cross-linked structure of cell wall composition critically hindered the saccharification hydrolysis in the ethanol production process. Genetic engineering had been reportedly successful in modifying cell wall compositions and increase saccharification hydrolysis efficiency by reducing cellulose content and crystallinity [48], decreasing xylan content [49], lowering lignin content [50] or altering lignin construction [51]. Additionally, the dynamic, adjustable, yet rigid cell wall structure dictates cell expansion. Changing of the cell size and shape is concerned with the alteration of cell wall composition or structure. Here, we reported that overexpression of OsPIL1 significantly improved the saccharification hydrolysis efficiency of transgenic switchgrass plants (TG11 and TG29) with or without pretreatment. The transgenic lines with lower expression of OsPIL1 showed no significant differences in the saccharification hydrolysis efficiency with WT plants, which suggested the high expression level of OsPIL1 was important to the saccharification hydrolysis efficiency. Higher expression of OsPIL1 was found to correlate with the longer cell length, which could be accompanied by severely changed cross-linked structure of cell walls, and then increased the accessibility of cellulose to cellulase enzymes during the saccharification process. Expansin S1 and Extensin protein-like are candidates of OsPIL1 downstream regulators. They are cell wall related genes responsible for cell wall loosening in structure, were reported to be up-regulated in rice overexpressing OsPIL1 [29]. HB2 (Homeobox Protein 2) is a target gene of PIF4, which is related to cell elongation and was found to be upregulated in the overexpressed OsPIL1 transgenic Arabidopsis thaliana [30]. Similarly, we also found that the expression of these three genes was up-regulated in OsPIL1 transgenic switchgrass. The results suggested that OsPIL1 enhanced switchgrass stem epidermal cells elongation and changed the cell wall structure hence improved saccharification hydrolysis efficiency via regulation of these genes. An OsPIL1 downstream candidate gene 1-aminocyclopropane-1-carboxylate (1-ACC) oxidase that participates in the biosynthesis of ethylene was up-regulated in TG plants, which suggested OsPIL1 might be modulate the ethylene signal pathway to regulate the growth of switchgrass like PIF5 did in Arabidopsis thaliana [41]. As the model plant of second-generation bioenergy crop, breaking its inherent recalcitrance of biomass and improving its saccharification hydrolysis efficiency is crucial to improve economic effectiveness. Improvement of saccharification hydrolysis efficiency by regulating relevant genes in the lignin biosynthesis pathway by genetic manipulation has been proved successful. For example, down-regulating the expression of 4CL [16], CAD [17] and COMT [18] in switchgrass reduced lignin content and improved saccharification hydrolysis efficiency. However, a significant reduction of lignin level of plants could affect its normal growth and stress tolerance [52]. Hence, it is necessary to search for other molecular tools to improve saccharification hydrolysis efficiency of switchgrass. On the other hand, although switchgrass itself is a kind of bioenergy plant with high biomass yield, but it is still necessary to further improve its biomass yield owing to the low bioconversion efficiency in the ethanol production process and limited land for cultivation. In fact, increasing both biomass yield and saccharification hydrolysis efficiency of feedstock at the same time has

alkaline pretreatment. As shown in Fig. 4A, there were no significant differences between TG and WT plants in carbohydrate composition (glucose and xylose) of DMS after sulfuric acid treatment. Intriguingly, we found that overexpression of OsPIL1 had a significant impact on saccharification efficiency of most transgenic plants without pretreatment. Transgenic plants that showed higher OsPIL1 expression level (TG12, TG11 and TG29) released more soluble sugar (glucose and xylose) and possessed higher saccharification efficiency than WT plant by direct enzymatic hydrolysis (Fig. 4B and C). The released glucose content of TG plants (58.32–63.89 mg/g DMS) had a 24.2%–36.1% increase compared to WT plant (46.95 mg/g DMS), resulting in remarkable differences in total sugar yield among them. However, there were no significant differences in releasing xylose yield between TG and WT plants (Fig. 4B). Saccharification efficiency of TG plants without pretreatment reached about 13%, which was significantly higher than that of WT plant (about 11%) (Fig. 4C). More sugar was released after alkaline pretreatment, TG11 and TG29 with higher expression level of OsPIL1 showed significant increase in released soluble sugar yield (Fig. 4D) and saccharification efficiency (Fig. 4E). We also analyzed the sugar release yield with enzymatic hydrolysis from one mature tiller (R3 stage) of WT and TG plants before (unpretreated) and after pretreatment. For the unpretreated DMS samples, excluding TG9, other TG plants produced significantly more soluble sugar (0.62-0.84 g/tiller) in subsequent fermentation compared to WT plant (0.45 g/tiller) (Fig. 4F). After alkaline pretreatment, about 100% more soluble sugar was produced compared to direct enzymatic hydrolysis. Among them, TG13, TG11 and TG29 produced significantly more soluble sugar (1.45–2.02 g/tiller) than WT plant (1.04 g/tiller) (Fig. 4F). The results indicated that overexpression of OsPIL1 in switchgrass plants improved saccharification efficiency of switchgrass TG plant, which is promising for a biofuel crop. 3.5. Overexpression of OsPIL1 enhanced expression of its downstream genes We tested the relative expression of downstream four candidate genes of OsPIL1 as mentioned in pervious report [29,30], including Expansin S1 (Pavir.J13116.1), Extensin protein-like (Pavir.Ib04078.1), 1aminocyclopropane-1-carboxylate (1-ACC) oxidase (Pavir.J25986.1) and Homeobox Protein 2 (Pavir.Gb01134.1). As shown in Fig. 5, the relative expression level of these four genes increased in the OsPIL1 overexpressing plants compared to WT plant. The result suggested that heterogenous expressing OsPIL1 in switchgrass affected the expression of downstream candidate genes of PIL1 and then promoted the growth of transgenic switchgrass. 4. Discussion Compared to food crops such as rice, wheat and corn that are paid much attention to the increase of grain yield and likely to be cultivated as dwarf plants, however the herbaceous energy plants are particularly emphasized on the improvement of plant biomass. High biomass yield not only provides more raw materials for bioethanol production, but also saves arable land, manpower and economic input. Plant height is a key factor affecting plant biomass. In this report, we improved the plant height and biomass yield of switchgrass plant by overexpression of an OsPIL1 gene. PHYTOCHROME-INTERACTING FACTOR LIKE (PIFs/ PILs) transcription factors reportedly participated in multiple signaling pathways, such as light [21–24], hormones [41,42], elevated temperature [43] and sucrose [44]. PIFs are important regulatory factors in plant development and abiotic stress. Recently, a maize (Zea mays) PIF transcription factor, ZmPIF1 reportedly played an important role in the ABA-mediated regulation of stomatal closure to control water loss, and was able to increase the grain yield through an increase in tiller and panicle numbers in transgenic rice [45]. Arabidopsis thaliana PIF4 and PIF5 are phylogenetically the closest orthologs of rice PHYTOCHROME-INTERACTING FACTOR 3-LIKE1 gene (OsPIL1) [28]. 149

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been reported. Down-regulating PdGAUT12.1 in Populus deltoids increased plant height, stems diameter and improved saccharification hydrolysis efficiency [49]. Overexpression of an ERF gene PvERF001 in switchgrass enhanced biomass yield and sugar releasing efficiency [53]. Transgenic switchgrass plants overexpressing miR156 increased biomass yield by enhancing tiller numbers and improved saccharification hydrolysis efficiency [13]. Wang et al reported that using STF in switchgrass resulted in improved biomass yield and saccharification hydrolysis efficiency [54]. In this report, we demonstrated that overexpression of OsPIL1 had no significant effect on the lignin content, but promoted biomass yield and saccharification hydrolysis efficiency of the transgenic switchgrass plants. Switchgrass can not only be used as a bioenergy crop, but also as a forage crop [55]. The forage quality of OsPIL1 transgenic switchgrass plants was higher than that of WT plant. With improvement on biomass yield, increase saccharification hydrolysis efficiency and high forage quality, the OsPIL1 transgenic switchgrass plants will offer more flexibility for farmers. In summary, heterologous expression of OsPIL1 in switchgrass promoted stem epidermal cell elongation, increased internode length, plant height and biomass yield. The saccharification hydrolysis efficiency of OsPIL1 transgenic plants was also improved. Thus, overexpression of OsPIL1 in switchgrass is an alternative approach to improve the economic value of bio-ethanol production with switchgrass as a feedstock.

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