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ABSTRACT Transgenic switchgrass overexpressing _Lolium perenne_ L. delta1-pyrroline 5-carboxylate synthase (_LpP5CS_) in group I (TG4 and TG6 line) and group II (TG1 and TG2 line) had

significant P5CS and ProDH enzyme activities, with group I plants (TG4 and TG6) having higher P5CS and lower ProDH enzyme activity, while group II plants had higher ProDH and lower P5CS

enzyme activity. We found group II transgenic plants showed stunted growth, and the changed proline content in overexpressing transgenic plants may influence the growth and development in

switchgrass. RNA-seq analysis showed that KEGG enrichment included phenylpropanoid biosynthesis pathway among group I, group II and WT plants, and the expression levels of genes related to

lignin biosynthesis were significantly up-regulated in group II. We also found that lignin content in group II transgenic plants was higher than that in group I and WT plants, suggesting

that increased lignin content may suppress switchgrass growth and development. This study uncover that proline can appropriately reduce lignin biosynthesis to improve switchgrass growth and

development. Therefore, appropriate reduction in lignin content and increase in biomass are important for bioenergy crop to lower processing costs for biomass fermentation-derived fuels.

SIMILAR CONTENT BEING VIEWED BY OTHERS OVEREXPRESSION OF _PVWOX3A_ IN SWITCHGRASS PROMOTES STEM DEVELOPMENT AND INCREASES PLANT HEIGHT Article Open access 01 December 2021 THE REGULATION OF

CELL WALL LIGNIFICATION AND LIGNIN BIOSYNTHESIS DURING PIGMENTATION OF WINTER JUJUBE Article Open access 01 November 2021 REGULATION OF PHENYLPROPANOID BIOSYNTHESIS BY MDMYB88 AND MDMYB124

CONTRIBUTES TO PATHOGEN AND DROUGHT RESISTANCE IN APPLE Article Open access 01 July 2020 INTRODUCTION Proline is known as a compatible solute (osmolyte) and a scavenger of reactive oxygen

species (ROS) providing protection against oxidative damage1. In addition to its well established role in coping with environmental stress, proline also plays an increasingly significant

role in plant development. It was reported that proline might have certain regulatory functions during protein synthesis and may act as a signaling molecule during plant development1, and

proline plays an important role in plant growth and life cycle, such as regulating cyclin genes and affecting general protein synthesis2. Additionally, proline may also play critical roles

in cellular metabolism both as a component of proteins and as a free amino acid3. Many genes are involved in the proline synthesis and degradation pathways. In higher plants,

Δ1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) are rate-limiting enzymes during the synthesis and degradation of proline respectively. Glutamate is reduced to

pyrroline-5-carboxylate (P5C) by P5CS, which is then converted into proline by Δ1-pyrroline-5-carboxylate reductase (P5CR)1. On the other hand, proline degradation takes place in the

mitochondria, where it is catalyzed into glutamate by ProDH and P5C dehydrogenase (P5CDH)4. Proline metabolism occupies a central place in plant metabolism and is connected to other pathways

through both ornithine and glutamate. It is connected to the pentose phosphate pathway and the TCA cycle as a way of moving the reductants and buffering the redox status of the

chloroplast4,5. In the proline biosynthesis pathway, the consumption of the reductants (NADPH) buffers the redox status of the chloroplast, which is linked with the pentose phosphate

pathway. In the pentose phosphate pathway, glucose-6-phosphate (G-6-P) is reduced to ribulose-5-phosphate (Ru-5-P) by the rate-limiting enzymes glucose 6-phosphate dehydrogenase (G6PDH) and

6-phosphogluconate dehydrogenase (6PGDH), and concurrently consumes NADP+ to generate NADPH in this process6. In plants, transketolase (TK) plays a role in Calvin cycle while in

non-photosynthetic organisms it connects the phosphate pentose pathway and glycolysis for generating NADPH7. In addition, proline degradation contributes carbon for TCA cycle4. The pentose

phosphate pathway and Calvin cycle provide erythrose-4-phosphate (E4P) which together with phosphoenolpyruvate acts as precursor for phenylalanine biosynthesis through the shikimic acid

pathway5. Phenylalanine, as the precursor amino acid for lignin biosynthesis, is essential for secondary cell wall biosynthesis8. In most plants, lignin is mainly composed of hydroxyphenyl

(H), guaiacyl (G) and syringyl (S) monolignol subunits that are derived from _p_-coumaryl, coniferyl and sinapyl monolignols, respectively. Several enzymes are required for monolignol

biosynthesis, including phenylalanine ammonia-lyase (PAL), caffeic acid 3-Omethyltransferase (COMT), caffeoyl-CoA O-methyltransferase (CCoAOMT), hydroxycinnamoyl-CoA reductase (CCR),

cinnamyl alcohol dehydrogenase (CAD)9. A number of reports have showed that lignin content is reduced by down-regulating the expression of monolignol biosynthesis related genes in plants10.

Many studies had showed that lignin content had the negative relationship with the growth and development of plants. Transgenic aspen with suppressed _Pt4CL1_ expression exhibited up to a

45% reduction of lignin and a 15% increase in cellulose, but leaf, root, and stem growth were substantially enhanced11. Additionally, compared with WT plants, _AtLOV1_ transgenic switchgrass

plants had higher total lignin content, delayed flowering time and less aboveground biomass12. Besides, a number of studies suggested that the most severe deficiency of lignin showed

stunted growth in plants13. However, many studies also showed that lignin can be reduced without reducing yield or fitness14,15. Switchgrass (_Panicum virgatum_ L.) is a perennial C4 grass

native to North America, considered as a potential dedicated bioenergy crop due to its high biomass production and tolerance on marginal land9. We found that overexpression plants showed two

different phenotypes, the two groups transgenic plants had different P5CS and ProDH enzyme activities, as well as proline content. To shed light on potential changes in growth and

development, we analyzed the RNA-seq data. The results showed that phenylpropanoid biosynthesis pathway was significantly up-regulated in the group II plants compared with the group I and WT

plants. In particular, lignin content in group II transgenic plants was higher than that in group I and WT plants, suggesting that proline affects switchgrass growth and development by

coordination with lignin biosynthesis. RESULTS P5CS AND PRODH ENZYME ACTIVITIES IN THE TWO GROUPS OF TRANSGENIC PLANTS P5CS and ProDH are the rate-limiting enzymes in the proline synthesis

and degradation pathway, respectively1. P5CS and ProDH activities showed significant differences among the group I (TG4 and TG6), group II (TG1 and TG2) and WT plants. P5CS activity was

lower in group II and higher in group I as compared to the wild type (Fig. 1A) where the P5CS activity in group I plants was 4.1 and 2.1 fold greater than group II and WT plants

respectively. On the contrary, ProDH activity was the highest in the group II plants (142 U/g), followed by the WT (124 U/g), and lowest in the group I plants (102 U/g) (Fig. 1B).

Additionally, group II overexpression lines showed relatively lower proline content, and the proline content of group I transgenic plants was higher compared with WT plants16. Combined with

the proline levels, these results suggested that proline synthesis is reduced and proline degradation is enhanced in the group II plants compared with the WT plants, and while in group I,

there is an increased proline synthesis and decreased proline degradation. COENZYME II NADP(H) CONTENTS IN THE TWO GROUPS OF TRANSGENIC PLANTS Group II transgenic plants contain lower

proline content, higher ProDH and lower P5CS enzyme activity compared with the WT plants. NADP+ and NADPH are generated during the synthesis and degradation of proline, respectively; proline

synthesis consumes NADPH to regenerate NADP+ in the chloroplast, which indicates the NADPH/NADP+ ratio is an important index for proline synthesis17. The coenzyme II NADP(H) content and

NADPH/NADP+ ratio was significantly different between the two groups of overexpression lines and WT plants (Fig. 2). The coenzyme II NADP+ content was the highest in group I plants, followed

by the WT, and lowest in group II plants. Conversely, coenzyme II NADPH was the highest in group II plants, 2.0- and 2.6-fold greater than that in WT and group I plants, respectively.

Additionally, the NADPH/NADP+ ratio was higher in group II plants and lower the group I plants compared with WT plants. These results suggested that the change in P5CS and ProDH enzyme

activities may result in the difference of coenzyme II NADP(H) contents in the group I, group II and WT plants, and simultaneously changing the proline level. MORPHOLOGICAL CHARACTERISTIC IN

THE TWO GROUPS OF TRANSGENIC PLANTS As shown in the Fig. 3A,B, group II plants exhibited more stunted growth, such as slender stem and fewer tillers. Compared to the WT plants, transgenic

plants in group II had a 52% and 23% reduction in tiller number and internode diameter, respectively; while transgenic plants in group I had a 48% and 19% increase in tiller number and

internode diameter, respectively (Fig. 3C,D). RNA-SEQ ANALYSIS In the RNA-seq data, the top 20 obviously enriched KEGG pathways are shown in Supplementary Fig. S1. In group II and group I

transgenic plants, these significantly enriched pathways include “Phenylpropanoid biosynthesis (KO: zma00940)”, “Stilbenoid, diarylheptanoid and gingerol biosynthesis (KO: zma00945)”,

“Cyanoamino acid metabolism (KO: zma00460)”, “Flavonoid biosynthesis (KO: zma00941)” and “Biosynthesis of secondary metabolites (KO: zma00564)”. Additionally, 13 pathways were significantly

enriched in the group II and WT plants. These include “Phenylpropanoid biosynthesis (KO: zma00940)”, “Biosynthesis of secondary metabolites (KO: zma01110)”, “Glutathione metabolism (KO:

zma00480)”, “Cysteine and methionine metabolism (KO: zma00270)”, “Stilbenoid, diarylheptanoid and gingerol biosynthesis (KO: zma00945)”, “Pentose phosphate pathway (KO: zma00030)”,

“Cyanoamino acid metabolism (KO: zma00460)”, “Flavonoid biosynthesis (KO: zma00941)”, “Selenocompound metabolism (KO: zma00450)”, “Nitrogen metabolism (KO: zma00910)”, “Biosynthesis of amino

acids (KO: zma01230)”, “Carbon metabolism (KO: zma01200)” and “Alanine, aspartate and glutamate metabolism (KO: zma00250)”. Finally, ten pathways were found significantly enriched which

include “Fatty acid degradation (KO: zma00071)”, “Tyrosine metabolism (KO: zma00350)”, “Selenocompound metabolism (KO: zma00450)”, “Isoquinoline alkaloid biosynthesis (KO: zma00950)”,

“Starch and sucrose metabolism (KO: zma00500)”, “Biosynthesis of secondary metabolites (KO: zma01110)”, “Tropane, piperidine and pyridine alkaloid biosynthesis (KO: zma00960)”, “Fatty acid

metabolism (KO: zma01212)”, “Peroxisome (KO: zma04146)”, “Carbon metabolism (KO: zma01200)”. Besides, the enriched KEGG pathways also included the photosynthesis and circadian rhythm-plant

pathways (Supplementary Fig. S2), suggesting that proline metabolism affects the photosynthesis and circadian rhythm in switchgrass. Notably, “Phenylpropanoid biosynthesis” pathway was

significantly enriched (_P_-value < 0.05). The expression levels of those DEGs including _PvPAL_, _PvCOMT_, _PvCCoAOMT_, _PvCYP98A3_, _PvCCR_, _PvCAD, PvG6PDH_, _Pv6PGDH_ and _PvTK_ (Fig.

 4), were all significantly up-regulated in the group II plants. Especially, the expression level of _PvCCR_ in group II was 12-fold greater than in the WT. In summary, the metabolic network

of enriched pathways included arginne and proline metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis and pentose phosphate pathway, indicating that proline metabolism is

involved in those metabolism pathways in switchgrass (Supplementary Fig. S3). LIGNIN MONOMER COMPOSITIONS IN THE TWO GROUPS OF TRANSGENIC PLANTS In the data of RNA-seq, the expression level

of differentially expressed genes (DEGs) involved in the phenylpropanoid biosynthesis were significantly up-regulated in the group II plants compared with the group I and WT plants.

Moreover, the lignin monomer composition in the two groups of transgenic and WT plants (Table 1), showed a relatively lower guaiacyl (G) and syringyl (S) contents, as well as hydroxyphnyl

(H) content in group I plants. While group II transgenic plants showed relatively higher guaiacyl (G) and syringyl (S) contents, as well as lower hydroxyphenyl (H) content compared with the

WT plants. Notably, the ratio of S/G was lower in the group I plants, and the lignin content (S + G + H monomer content) showed a significant difference between the two groups of transgenic

and WT plants. Lignin content was lower in the group I plants and higher in the group II plants compared with the WT. In addition, S monomer content of the stem from TG2, TG6 and WT at the

R1 stage was evaluated after staining with the Mäule reagent, we found that the S monomer content (Mäule staining) was clearly reduced in the stem of TG6 (Fig. 5). These results suggest that

proline metabolism affects lignin biosynthesis in switchgrass. DISCUSSION P5CS activity was lower in group II plants, which could led to the decreased proline accumulation. In the present

study, KEGG enrichment included pentose phosphate pathway (PPP), fatty acid synthesis and phenylalanine biosynthesis pathway, and at the same time, NADP+ and NADPH content showed a

significant difference between the two groups of transgenic and WT plants. NADP+ and NADPH are generated during the synthesis and degradation of proline, respectively18. The cycling of

proline substrate is coupled to maintaining the NADP+/NADPH ratio via the pentose phosphate pathway19, and PPP provides NADPH for fatty acid synthesis in plastids6. So it seems that proline

degradation was accelerated in the group II transgenic plants, which led to an increase in the NADPH content. It was reported that antisense _AtP5CS_ transgenic Arabidopsis led to proline

depletion and abnormal leaf formation20. Down-regulation of proline biosynthesis genes, on the other hand, resulted in growth defects21, indicating the importance of proline for plant

development. In our study, the stems were significantly thicker in group I transgenic plants and thinner in group II plants, when compared with the WT plants (Fig. 3D). Furthermore, as the

results of the RNA-seq analysis indicated, the enriched KEGG pathways also include the photosynthesis and circadian rhythm-plant pathways (Supplementary Fig. S2). This leads us to conclude

that proline may play important roles in plant growth and development. Proline metabolism is connected to the pentose phosphate pathway (PPP) and the TCA cycle, and the PPP and Calvin cycle

provide erythrose-4-phosphate (E4P) which together with phosphoenolpyruvate acts as a precursor for phenylalanine biosynthesis through the shikimic acid pathway5. In our result of RNA-seq,

the enriched KEGG pathways were including arginine and proline metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis and pentose phosphate pathway (Supplementary Fig. S3). To

precisely understand the relationship between proline metabolism and lignin biosynthesis in switchgrass, we summarized the metabolic network that exists between the phenylpropanoid

biosynthesis pathway, pentose phosphate pathway and proline metabolic pathway in Fig. 6. In these metabolism pathways, _PvG6PDH_ and _Pv6PGDH_ are genes encoding key enzymes involved in the

pentose phosphate pathway; _PvPAL_, _PvCOMT_, _PvCCoAOMT_, _PvCCR_ and _PvCAD_ are the genes encoding for important enzymes in lignin biosynthesis, and transketolase (TK) participates in the

Calvin cycle. The proline biosynthesis pathway is linked with the pentose phosphate pathway through consuming the reductants (NADPH), while the pentose phosphate pathway and the Calvin

cycle provide erythrose-4-phosphate (E4P) for phenylalanine biosynthesis. Thus, proline metabolism pathway is coordinated with phenylpropanoid biosynthesis pathways in switchgrass. In

particular, our results showed that the lignin content (S + G + H monomer content) was lower in group I plants and higher in group II plants compared with the WT. So, reducing proline

biosynthesis may induce the increase of lignin content in group II transgenic plants. Many studies showed that lignin content affected plant growth and development. Suppressing _Pt4CL1_

expression can reduce the lignin content and enhance the growth of leaf, root, and stem in aspen11, and RNA interference of _Pv4CL1_ lead to a reduction in lignin content with uncompromised

biomass yields15. Additionally, overexpressing _AtLOV1_ gene increased total lignin content, delayed flowering time and reduce the aboveground biomass in switchgrass12. Besides, extreme

lignin deficiency can also contribute to stunted growth in plants13. In our result, group II transgenic plants which had lower proline and higher lignin content exhibited more stunted

growth. Thus, we speculate that proline affects switchgrass growth and development by coordination with lignin biosynthesis. In conclusion, unlike the previous studies that proline plays an

important role in regulating cyclin genes and affecting general protein synthesis to improve plant growth and development. Our study showed that lacking proline could result in an increased

lignin content, and leading to the stunted growth in switchgrass. However, the regulatory mechanism how proline plays roles in plant growth and development is still unclear, and

understanding the molecular mechanism will help us to prime candidates in crop genetic engineering for improving the biomass of plants. Moreover, appropriate reduction in lignin content and

increase in biomass is one important strategy of bioenergy crop to lower processing costs for biomass fermentation-derived fuels. MATERIALS AND METHODS PLANT MATERIALS AND GROWTH CONDITIONS

Switchgrass callus generated from mature seeds (Alamo) was transformed with a pCAMBIA 1301-_LpP5CS_ overexpression cassette using the Agrobacterium-mediated transformation method16. Group I

consists of two independent overexpression lines (TG4 and TG6) while group II contains TG1 and TG2. The two groups of transgenic switchgrass plants were grown in the greenhouse under a 16 h

light/8 h dark photoperiod at 25 ± 2 °C. The switchgrass plants were used for the measurement of tiller number and stem diameter at reproductive stage (R3), and each line comprises three

biological samples. P5CS AND PRODH ENZYME ACTIVITIES P5CS and ProDH activities in the transgenic and control plants were assayed based on the protocal22,23. Briefly, frozen switchgrass

leaves (0.1 g) were ground in liquid nitrogen and mixed with 1 mL of extraction buffer (0.1 M Tris–HCl, pH 7.2, 10 mM MgCl2, 10 mM 2-mercaptoethanol, and 1 mM PMSF). The homogenate was

centrifuged at 12,000 g for 20 min at 4 °C and the P5CS activity was calculated. Switchgrass leaves (0.1 g) were homogenized into powder and placed in 0.5 mL extraction buffer (100 mM sodium

phosphate, 1 mM cysteine, and 0.1 mM EDTA [pH 8.0]). After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was used to measure ProDH activity. Each assay included two

technical with three biological replicates. COENZYME II NADP (H) CONTENTS The coenzyme II NADP(H) contents in the two groups of overexpression lines and WT plants were measured according to

the protocol24,25 with some modifications. Briefly, swithgrass leaves (0.1 g) were ground in liquid nitrogen and homogenized with 0.1 M HCl (for NADP assay) or 0.1 M NaOH (for NADPH assay).

Samples were heated at 95 °C for 2 min and cooled in an ice bath. After samples were centrifuged for 10 min at 4 °C, and the supernatants were used for coenzyme assay. Assays were performed

using a reaction volume of 200 mL on 96-well plates, and absorbance was measured at 565 nm from 0 to 30 min after the start of the reaction. Two technical and three biological replicates of

each transgenic line were performed in each experiment. RNA-SEQ ANALYSIS Total RNA was extracted from transgenic and WT leaves at the E5 stage, and RNA samples included three groups: TG1 and

TG2 lines (group II), TG4 and TG6 lines (group I), and WT1 and WT2 plants. The RNA-seq data were downloaded from NCBI, the SRA number is SRP13027516. The paired-end reads were aligned to

the reference genome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvirgatum_er) using TopHat v2.0.1226. The HTSeq v0.6.127 and DEGSeq R package28 were used to count the

reads numbers mapped to each gene and determine if any differential expression exists. The KOBAS software was performed to test the statistical enrichment of differential expression genes in

KEGG pathways. QUANTITATIVE REAL-TIME RT-PCR ANALYSIS Total RNA from the leaves of the transgenic and WT plants at elongation stage (E5) was isolated by the TRIzol reagent method

(Invitrogen, Carlsbad, CA, USA). Quantitative real-time RT-PCR analysis was performed following the description17, the data were normalized using the level of switchgrass _Ubq_ transcripts

(GenBank accession no. FL955474.1), and the relative expression levels of genes were calculated using the 2−△△CT method29. Two biological with three technical replicates of each line were

performed in each experiment. The primers used for qRT-PCR are listed in Table S1. LIGNIN MONOMER COMPOSITION Lignin monomer levels of stems from the two groups of transgenic and WT lines at

the reproductive stage (R1) were measured following the description30. Three technical replicates of each line were performed in the experiment. HISTOLOGY AND MICROSCOPY The internode 1

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Data from each trait were subjected to analysis of variance (ANOVA). The significance of the difference between treatments was tested at the _P_ < 0.05 level. Standard errors are provided

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(2004). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS Funding for this work was provided by the Ministry of Science And Technology, China (2014BAD23B03), National

Natural Science Foundation of China (31672478) and Natural Science Foundation of Beijing (6162016). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * College of Grassland Science and Technology,

China Agricultural University, Beijing, China Cong Guan, Hui-Fang Cen, Xin Cui, Dan-Yang Tian & Yun-Wei Zhang * Beijing Key Laboratory for Grassland Science, China Agricultural

University, Beijing, China Yun-Wei Zhang * National Energy R&D Center for Biomass (NECB), Beijing, China Yun-Wei Zhang * Beijing Sure Academy of Biosciences, Beijing, China Yun-Wei Zhang

* Department of Plant and Soil Sciences, Institute for Agricultural Bioscience, Oklahoma State University, Oklahoma, OK, USA Dimiru Tadesse Authors * Cong Guan View author publications You

can also search for this author inPubMed Google Scholar * Hui-Fang Cen View author publications You can also search for this author inPubMed Google Scholar * Xin Cui View author publications

You can also search for this author inPubMed Google Scholar * Dan-Yang Tian View author publications You can also search for this author inPubMed Google Scholar * Dimiru Tadesse View author

publications You can also search for this author inPubMed Google Scholar * Yun-Wei Zhang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

Conceived and designed the experiments: Y.Z. Performed the experiments: C.G., H.C., X.C. and D.T. Analyzed the data: C.G. and Y.Z. Wrote the paper: C.G. and Y.Z. Edited language: T.D. All

authors reviewed and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Yun-Wei Zhang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

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CITE THIS ARTICLE Guan, C., Cen, HF., Cui, X. _et al._ Proline improves switchgrass growth and development by reduced lignin biosynthesis. _Sci Rep_ 9, 20117 (2019).

https://doi.org/10.1038/s41598-019-56575-9 Download citation * Received: 02 October 2019 * Accepted: 08 December 2019 * Published: 27 December 2019 * DOI:

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