Impact of Expressing p-Coumaryl Transferase in Medicago sativa L. on Cell Wall Chemistry and Digestibility

The addition of p-coumaric acid (pCA) to lignin molecules is frequently found in members of the grass family. The role of this addition is not clearly understood, but is thought to potentially aid in the formation of syringyl-type lignin. This is because the incorporation is as a conjugate of pCA ester linked to sinapyl alcohol, a major component of lignin. The forage legume alfalfa (Medicago sativa L.) does not contain appreciable levels of pCA in its more heavily lignified stem tissues. The maize pcoumaryltransferase (pCAT) gene was used to transform alfalfa to determine its impact upon lignin composition and its potential to alter cell wall digestibility. A constitutive expression vector using the cassava vein mosaic virus (CsVMV) promoter was used to drive expression of maize pCAT in alfalfa. Expression of the pCAT transgene was detected in both leaves and stems. Though there was a range of pCAconcentration in transformed alfalfa stems (0.2 1.79 micrograms (μg)), this was a clear increase over bound pCA in control stems (0.15 0.2 mean = 0.17 micrograms (μg)). This did not lead to consistent responses concerning total lignin in the stem tissues. Leaf tissue, on the other hand, already has a relatively high level of pCA (0.85 1.2, mean = 0.99 micrograms (μg)) and those expressing pCAT gene showed on average a small increase, but there is a wide range of values among the transformants (0.38 1.55, mean = 1.06 micrograms (μg)). Lignin in leaves did not appear to be significantly impacted. However, incorporation of pCA into the wall appears to cause a shift in lignin composition. Testing the pCAT expressing stem cell walls for digestibility using a rumen in vitro system showed there was no change in the digestibility of the stem compared to empty vectors and control alfalfa stems. Although expression of pCAT gene in alfalfa changes the amount of wall bound pCA, it does not appear to change lignin levels or impact digestibility. *Mention of a trademark or proprietary product does not constitute a guarantee or warranty of product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. How to cite this paper: Marita, J.M., Rancour, D., Hatfield, R. and Weimer, P. (2016) Impact of Expressing p-Coumaryl Transferase in Medicago sativa L. on Cell Wall Chemistry and Digestibility. American Journal of Plant Sciences, 7, 2553-2569. http://dx.doi.org/10.4236/ajps.2016.717221 Received: November 16, 2016 Accepted: December 27, 2016 Published: December 30, 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Phenolic compounds are ubiquitous among higher-order plants, primarily existing as lignin, a polymer composed of the monolignols p-coumaroyl, coniferyl, and sinapyl alcohols [1] [2]. Simpler forms of phenolic compounds are also quite prominent in a wide range of plants, and are known to have antioxidant roles and health benefits in human diets [3] [4] [5]. Compounds like methylcinnamate and methyl p-coumarate are often found as components in floral scents [6]. Grasses have both complex phenolic polymers (lignin) and monomeric phenolic compounds incorporated into their cell walls [7]. Ferulates in grass cell walls have been well investigated and their roles within plants are well established [8] [9] [10]. The p-coumarates (pCA) found in grass cell walls have also been studied but their role has not been well defined. Incorporation of pCA into cell wall matrices can occur in two forms: one as an ester linked conjugate to arabinofuranose (Araf) of glucuronoarabinoxylans (GAX) [11] and secondly as an ester linked conjugate of pCA and sinapyl alcohol [12]. Only the alcohol (sinapyl or coniferyl alcohol) becomes incorporated into the developing lignin polymer leaving the p-coumarate portion to remain free and linked only by the original ester bond of the conjugate. A specific transferase has been identified that is responsible for the formation of the pCA-sinapyl alcohol conjugate (p-comuaroyl-CoA: sinapyl alcohol transferase, pCAT) [13] [14]. Recently a gene for p-coumaryl-CoA: sinapyl alcohol transferase has been identified from rice (Oryza sativa) and the expressed protein in an E. coli system showed a strong preference for p-coumaryl CoA and sinapyl alcohol as substrates [15].
It has been proposed the formation of p-coumaryl-sinapyl alcohol (pCA-SA) conjugates could aid in the incorporation of sinapyl alcohol into lignin resulting in what is referred to as syringyl-rich regions of lignin [16]. In corn (Zea mays L.) sinapyl alcohol is not easily oxidized by the cell wall peroxidase: H 2 O 2 system, but pCA is readily oxidized by the same system. The oxidized form of pCA (pCA radical) can be readily transferred to sinapyl alcohol residues creating a sinapyl alcohol radical that can readily undergo radical-mediated coupling reactions with other monolignol radicals [16] resulting in syringyl-rich lignin region. Whether this is the main function of pCA incorporation into grass cell walls remains to be determined.
Following up on work identifying a p-coumaroyltransferase (pCAT) in corn led to isolation and characterization of this enzyme and ultimately the identification of the putative gene for pCAT. An RNAi construct of the gene was produced and used to genetically modify corn. To test the hypothesis that pCA-SA could alter lignin composition, the gene was inserted and expressed in Medicago sativa L. Would this lead to an accumulation of pCA in Medicago cell walls and would this influence digesti-bility?

Plant Culture
Alfalfa was grown in the greenhouse (under high pressure sodium lamps, 14/10 day/ night regime, 25˚C -35˚C). Plants were harvested at the late bud to early flower stage of development, frozen in liquid nitrogen, and stored in a −80˚C freezer until analyzed.
Leaves and stems were stored separately.

Vector Cloning and Plant Transformation
The maize pCATcDNA expression vector was generated using Gateway® LR Clonase™ II enzyme mix (Life Technologies) as previously described [17] to recombine the cDNA-containing pDONR221-pCAT [13] and pMLS509 (aka pJCV86) [13], a constitutive expression vector using the cassava vein mosaic virus (CsVMV) promoter to con-

Transgenic Plant PCR Screening
Leaf genomic DNA (gDNA) was isolated based on the procedure of Rancour et al. [17].
Multiplexed PCR screening of plants for the neomycin phosphotransferase II plant-selection marker and a NADH-dependent glutamate synthase, a general gDNA marker, was performed using primers RH196, RH197, MS152, and MS153. Table 1 shows the primers used in this study.

Statistical Analysis
One-way ANOVA with a post hoc Tukey test was performed using GraphPad Prism software (version 5.0f). An alpha value of 0.05 was used for all analysis.

Cell Wall Analyses
An abbreviated method of cell wall extraction [25] was used to prepare samples for analysis. Samples were ground in a freezer mill (SPEX 6850) after a 3 min precool and three cycles of 1 min run time and 2 min cool time at a rate of 12 CPS (impacts per second). Milled samples were transferred to pre-weighed Oakridge tubes cooled in liquid nitrogen and stored at −80˚C until further processing. Milled samples were extracted with 50 mM NaCl overnight at 4˚C followed by 30 min at 40˚C C the next morning. Material was pelleted by centrifugation at 22,000 x g (at average radius) for 20 min at 20˚C. Supernatants were decanted and pellets extracted 2 times more with 50 mM NaCl for 30 min at 40˚C. Pelleted material was suspended in 50 mM Tris-Acetate pH 6.5 and heated for 2 h in boiling water bath to gelatinize starch. Samples were cooled (~55˚C), treated with 40 U amyloglucosidase (Fluka Bio Chemika) and 20 U of 1,4-α-D-glucan glucanohydrolase (α-amylase; Sigma-Aldrich, St. Louis, MO), then incubated at 55˚C for 2 h with shaking. Reactions were terminated by adding ethanol (95%) to a final concentration of 80% and mixing at room temperature for 30 min. Samples were centrifuged as above, supernatants aspirated off and EtOH extracts properly disposed. Pellets were extracted an additional 3 times with 80% EtOH, followed with acetone washes, after which samples were stored overnight at 4˚C. The next day samples were brought to room temperature and incubated with shaking for 30 min. Material was centrifuged as before and supernatants removed. Pellets were extracted 1 time with chloroform: methanol (1:1 v/v) followed by 4 extractions with acetone, with all extractions for 30 min at room temperature while shaking. Cell wall residues were air dried in a fume hood for 2 d to evaporate organic solvents. After air drying, cell walls were placed in an oven (55˚C) for 48 hours to fully dry, and weighed to determine yields. Cell wall material was stored dry at room temperature until further use.
Samples were maintained in an oven (55˚C) overnight before analyses. Dry samples were incubated 2 h at 50˚C in 2.5 ml 25% (v/v) acetyl bromide in glacial acetic acid.
Samples were cooled to room temperature and any insoluble material remaining was cleared using a microfuge (1.5 mL of sample, 3 min, 12,000× g). Clarified supernatant (0.5 ml) was transferred to a glass scintillation vial containing 9.5 ml of 0.42 M NaOH, 18.4 mM hydroxylamine, and 12.4 M acetic acid. Absorbance scans from 350 -250 nm were taken and the absorbance at 280 nm was used to calculate sample lignin content.
The extinction coefficients used for calculations were 15.302 g −1 L·cm −1 , for alfalfa cell wall samples and maize stock cell wall standard [28]. The extinction coefficient used for alfalfa samples was the average of values determined for alfalfa stems obtained from purified HCl-dioxane lignin preparations [28]. Duplicate cell wall preparations were analyzed and data were compiled according to transformation events.

Ester-and Ether-Linked Phenolics
Cell wall ester-and ether-linked phenolic moieties were determined using the sequential analysis as described [29].

Total Cell Wall Digestibility
Alfalfa stem digestibility was measured using the gas pressure method described by Weimer et al. [33]. Methods were modified to use 10 mL serum vials (calibrated as described by Weimer et al. [33]) and using a sampleof 20 mg accurately weighed to 0.01 mg into 10 mL serum vials. In vitro gas production was measured with a hand held where V = cumulative mL of gas (g of digestible organic matter) −1 ; A= asymptotic mL maximal gas production (g of digestible organic matter) −1 ; k= first-order rate constant; and After completion of each 96 hour digestion samples were analyzed for total VFA production by HPLC [34]. Total VFA concentrations were the sum of C2 to C5 strai-ght-and branched-chain VFA. The energy content of the VFA was calculated as total alkyl groups (i.e., total methyl and methylene groups within the VFA).

Results and Discussion
To address the functional role of pCA in plant cell walls, we chose to stably express the maize p-coumaroyl-CoA: hydroxycinnamyl alcohol transferase in alfalfa (Medicago sativa), an agronomically important crop plant that lacks lignin-associated endogenous ester-linked pCA. In principle, this experimental system can be viewed as a gain-of-  Alfalfa plant stems and leaves were analyzed separately for major cell wall components. Of particular interest was the impact upon pCA and lignin within cell walls of leaf and stem tissues. Within the stem cell wall fraction pCA was detectable at a background level of approximately 0.2 g·kg −1 CW. Variable levels of corn pCAT gene expression resulted in a wide variation in pCA incorporation into stem CW. Additional pCA incorporation above background varied from zero to nine times the background resulting in a significant increase in the ester linked pCA in stems compared to control plants (Figure 4). Although there were significant increases inpCA content the level was lower than what is seen in typical grass stem tissue. The pCA content of grass cell walls varies widely depending upon thespecies ranging from 5 g to 37 g·kg −1 CW [37].
Alfalfa leaf material on the other hand already contains relatively higher levels of pCA compared to the stems. It is not clear whether this pCA is ester linked to lignin or carbohydrates. Analysis of whole cell wall material by NMR [30] does not allow us to identify pCA in the cell wall due to the limited incorporated into the wall ( Figure 5).
However, when comparing the total levels in alfalfa leaves to grass leaf tissue, levels are not so different from grass leaf blade tissues [38].
This may not be too surprising because lignin levels are typically lower in leaf tissues compared to stems especially when considering the leaf tissue minus the midrib.
Therefore the metabolic processes producing the monolignols will be less active than in the stems, i.e., limiting the supply of sinapyl alcohol, the preferred acceptor for the pCAT [25] [37]. If the alfalfa leaf pCA is not associated with lignin at least in the same fashion as seen with grass lignins a possibility to consider is the incorporation of pCA into the carbohydrate fraction of leaf cell walls. Mild acid hydrolysis can be used to release arabinofuranose (Araf) residues from arabinoxylans. If pCA and/or FA are attached to the Araf residue they will remain intact and one can measure the pCA-Arafconjugate to determine the amount that is attached to at least this type of carbohydrate fraction. We have used this technique to successfully distinguish between pCA and FA bound to arabinoxylans in grasses [17]. However, this technique coupled with GC-MS analysis failed to show any pCA-Araf in alfalfa cell walls. The fact that the pCA is alkaline labile under relatively mild conditions most likely indicates it is ester linked within the wall matrix, but the wall component it is esterified to remains unclear at this time.It is possible that pCA is attached to ligninal though such attachment does not appear to  This assumes the conjugate is not ending up in lignin at least in large quantities.
We selected some of the stem tissues that showed changes in pCA content to test with an in vitro rumen fluid digestion assay. Total digestibility was measured as cumulative amount of gas produced over the 96 hours (Table 3) and by an analysis of total  Results averaged over four different replicate runs indicated there was no difference in any of these fermentation metrics between those expressing pCAT, and empty vectors as alfalfa control samples. This may indicate that pCA incorporation into the cell wall has no impact upon cell wall organization. Alternatively the changes induced within the plants by the addition of pCA conjugates were not sufficient to cause changes in cell wall structure and function, e.g., digestibility or that pCA-sinapyl alcohol conjugates do not influence total cell wall digestibility.

Conclusion
Modification of alfalfa by insertion of the maize pCAT gene resulted in increased amounts of p-coumarates in the cell walls. The pCAT gene expression levels were simi- lar between leaves and stems resulting in similar levels of p-coumartes. However, the leaves before transformation contain relatively high levels of pCA compared to stems. Actual increases in pCA were much more pronounced in the stem cell walls with increases as high as 8 to 9 times higher than the empty vector controls. Accumulation of pCA over empty vector controls in leaves was restricted to 20% to 60% increase in those plants that increased in pCA levels. The large changes in cell wall pCA in stems did not lead to changes in lignin accumulation or a detectable shift in composition. This change in stem pCA did not result in a change in cell wall digestibility. It is possible that even though there were changes in stem associated pCA, it was not sufficient to disrupt the normal lignification process and alter digestibility.