Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Biodegradation Profiles of Proanthocyanidin-Accumulating Alfalfa Plants Coexpressing Lc-bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes R. G. Heendeniya, M. Y. Gruber, Y. Lei, and Peiqiang Yu*
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Department of Animal and Poultry Science, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK Canada, S7N5A8 Saskatoon Research and Development Center, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK Canada S7N 0X2 ABSTRACT: The utilization of the nutrient potential of alfalfa (Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if proteinbinding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 μg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems. KEYWORDS: Lc-bHLH gene, C1-MYB gene, gene transformation, plant feed biotechnology, proanthocyanidins, nutrition and structure interaction, Medicago sativa L. plant
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INTRODUCTION Alfalfa (Medicago sativa L.), aka lucerne, is a highly nutritious, winter-hardy, high-yielding Leguminosae pasture crop.1,2 However, the utilization of alfalfa up to its nutritional potential is curtailed by the rapid rumen degradation of its protein that leads to protein waste3,4 and frothy bloat, particularly under grazing conditions.2,5 For more than three decades, researches have worked on developing a “bloat safe” alfalfa cultivar or a germplasm. In 1997, an alfalfa cultivar called AC Grazeland Br. was released that has shown reduced bloat incidences in grazing cattle.6 The thicker epidermal and mesophyll cell walls in this alfalfa cultivar contribute to lower incidences of bloat.5 The rate and extent of protein degradation as well as methane production in the rumen could be reduced if the protein in alfalfa is bound to condensed tannin (CT, aka proanthocyanidins), as demonstrated with legume plants naturally rich in these compounds.7−10 Jonker et al.11 reported that proanthocyanidin-accumulated alfalfa had reduced foaming properties, indicating the potential of proanthocyanidins in preventing rumen bloat. Tannin-bound proteins are degraded at a lower rate in the rumen and will be dissociated from tannin when they reach the acidic abomasum,7 where they become available © XXXX American Chemical Society
for enzymatic digestion and thereby increase the protein utilization efficiency.3,12 The transformation with transcription factors (TF) has been the most successful strategy for increasing the proanthocyanidin levels in aerial parts of the alfalfa. However, efforts with TFs, such as maize Lc-bHLH,13 Medicago trancatula LAP1-MYB, and Arabidopsis PAP1-MYB14 and clover TaMYB14,15 have resulted in varying degrees of success. The Lc-bHLH plants had a lower rate of N and DM degradation but were not winter-hardy,12,16 while LAP1-MYB transformation resulted in only proanthocyanidin-like compounds.14 Some of the initial plants transformed with TaMYB14 were reported to have proanthocyanidin contents ranging from 0.1 to 1%, but no further studies have been reported afterward. On the basis of the studies on model plants such as Arabidopsis thaliana, there are several families of transcription factors (i.e., bHLH, MYB, WD40, WIP-Zn finger, WRKY, and Received: January 25, 2019 Revised: March 7, 2019 Accepted: April 2, 2019
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DOI: 10.1021/acs.jafc.9b00495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry MADS) that are involved in the regulation of flavonoid pathways.17,18 They may act alone or in combination to regulate different stages of flavonoid pathways.19 In particular, the MYB-bHLH-WD40 protein complex positively regulates the latter stages in Arabidopsis flavonoid pathways.20,21 Because the single gene transformation of bHLH or MYB has not achieved the goal of increasing forage proanthocyanidins to moderate levels while maintaining alfalfa vitality, double gene transformation using two or three partners of the MYB-bHLHWD40 complex may potentially succeed in increasing proanthocyanidins or both in alfalfa. Alfalfa genotype transformed with both Lc-bHLH and maize C1-MYB became available to our research team. Our previous publication described the gene transformation, gene detection, and influences of such a genetic transformation on the inner molecular structures of alfalfa.22 In the current study, our specific objective was to investigate the influence of Lc-bHLH and C1-MYB gene transformation on proanthocyanidin accumulation, chemical composition, and protein degradability compared to the single gene transformed alfalfa (Lc-bHLH or C1-MYB), nontransgenic (NT) parent plants, and bloatreduced standard industry cultivar AC Grazeland Br (ACG).
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DNA insertions) that were growing robustly. All selected populations were transferred along with NT and ACG genotypes to a growth chamber where the light intensity, duration of light/dark, and temperature were maintained throughout the trial period at 550 μE m−2 S1−, 16h/8h, and 22 °C, respectively. Plants were randomly placed within the growth chamber, and the positions were changed randomly throughout the forage collection. The alfalfa forage was harvested continuously from all subsets of the original populations by shearing regrowth material 5 cm above the soil level, when the plants had reached late-bud stage.23 The harvested materials were immediately frozen at −20 °C and freeze-dried for storage at −20 °C. Once adequate amounts were collected from each plant, they were ground to pass through a 1 mm sieve (Retsch SM-3000, Brinkmann Instruments, ON, Canada) and stored at +10 °C. Analysis for Proanthocyanidins. Proanthocyanidin was extracted from freeze-dried and ground (1 mm) samples with 70% aqueous acetone according to Terrill et al.24 The extracts and solid residue were hydrolyzed with butanol-HCl (70/30%), and spectrophotometric absorbance was recorded at 550 nm using background subtraction. The proanthocyanidin contents were determined from a standard curve of dimeric commercial Procyanidin-B1 (epicatechin [4β-8] catechin) (Indofine Chemical Co Inc., Hillsborough, NJ, USA). Analyses for Chemical and Nutrient Profiles. Dried ground samples were analyzed according to AOAC25 for dry matter (DM; method 930.15), crude protein (CP; method 984.13), ash (method 924.05), ether extract (EE; AOAC 954.02), acid detergent fiber (ADF; method 973.18), and acid detergent lignin (ADL; method 973.18 followed by 72% H2SO4 treatment). The neutral detergent fiber (NDF) was determined according to the procedure proposed by Van Soest et al.26 The acid detergent insoluble crude protein (ADIP) and nonprotein nitrogen (NPN) were determined according to the procedure of Licitra et al.27 The α-amylase amyloglucosidase method (Megazyme Total starch Assay kit) was used to determine the starch content. The nonfiber carbohydrate (NFC) content was calculated as NFC = 100 − ash − CP − EE − (NDF-NDIP). The true protein (TP) content was calculated as TP = CP − NPN-ADIP.28 Protein and Carbohydrate Subfractions Related to Rumen Degradation and the Nutrient Supply Feature in Ruminant Systems. Protein subfractions and carbohydrate subfractions that are related to rumen degradation and the nutrient supply related to dairy cattle were determined by the updated Cornell Carbohydrate and Protein System (CNCPS).29 Briefly, CNCPS divides carbohydrate and protein pools into eight (CA1, CA2, CA3, CA4, CB1, CB2, CB3, and CC) and five (PA, PB1, PB2, PB3, and PC) subfractions, respectively.29 Within carbohydrate pools, CAs (organic acids and sugars), CB1 (starch), and CB2 (soluble fiber) are considered to be rapidly degraded, while CB3 (available NDF) is regarded as slowly degraded in the rumen. In protein pools, PA (NPN) and PB1 (soluble true protein) are regarded as rapidly degraded, while PB2 and PB3 are considered to be slowly degraded in the rumen. Both C fractions in carbohydrate pools and protein pools are considered to be unavailable fractions. In previous studies30,31 with HB12 and TT8 alfalfa, this method was applied to evaluate the gene silencing impact. Statistical Analyses. All data were analyzed by PROC MIXED of SAS32 version 9.3 according to statistical model Yij = μ + Pi + εij, where Yij is the dependent variable, μ is the overall mean, Pi is the fixed effect of alfalfa genotypic populations (i = 7; NT, ACG, C1, Lc1, Lc3, Lc1C1, and Lc3C1), and εij is the residual error. Tukey’s test was used for multiple population comparisons with letter groupings obtained using the SAS pdmix800 macro.33 The contrasts between means of different combinations of alfalfa populations were conducted using a contrast statement in SAS. For all statistical analyses, significance was declared at P < 0.05 and the tendency was declared at 0.05 < P < 0.10.
MATERIALS AND METHODS
Pre-experiment: Plant Material Development. Experimental populations of Lc1and Lc3 (both described in ref 1) and Lc1C1 and Lc3C1 (both developed by Dr. M. Y. Gruber, Agriculture and AgriFood Canada) and parental nontransgenic (NT) alfalfa were grown from seeds and maintained in a greenhouse at the Saskatoon Research Center (SRC), Agriculture and Agri-Food Canada. Each single transgene population was composed of progeny from previous transgenic plants crossed with a conventional alfalfa molecular breeding clone from Forage Genetics International, while the ditransgenic populations were developed from crosses between single transgene populations. Bloat-reduced alfalfa cultivar AC Grazeland Br. (ACG) was propagated vegetatively from existing plant stock maintained in the greenhouse. DNA (for genetic verification) was extracted from leaf samples collected from individual transgenic and NT genotypes using a DNA extraction kit (DNeasy Plant Mini Kit, Qiagen). After extraction, DNA samples were stored at −20 °C and used to amplify each respective transgene using PCR with the following specific primers.
Experimental Alfalfa Material and Gene Transformation for the Current Study. All of the plant materials were grown and maintained at the Saskatoon Research Center, Agriculture and AgriFood Canada under the guidance of Dr. M. Y. Gruber. The single gene (Lc1, Lc3, and C1) and double gene (Lc1C1 and Lc3C1) transformed alfalfa genotypes along with the NT plants were grown from seeds initially in the greenhouse. The nontransgenic ACG plants for this experiment were propagated from existing plant stock maintained at the greenhouse. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Subsets of genotypes (Lc1C1; Lc3C1; Lc1, Lc3, and C1) were selected from population genotypes confirmed with transgenes (or T-
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RESULTS AND DISCUSSION Effect of Gene Transformation on Proanthocyanidin Accumulation. Both the Lc single gene and LcC1 double B
DOI: 10.1021/acs.jafc.9b00495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Proanthocyanidins content in the present study was higher than that previously reported. Wang et al.16 reported lower proanthocyanidin contents with Lc transgenic alfalfa (Lc1, 114.6 μg/g of DM; Lc3, 102.2 μg/g of DM). Jonker et al.,1,34,39 who worked with Lc-transgenic alfalfa crosses progenies (with commercial alfalfa varieties), reported average proanthocyanidin contents from 163 to 232 μg/g of DM that varied between years of cultivation. The variation of proanthocyanidin accumulation among different experiments is likely influenced by the growing conditions.35 Effect of Gene Transformation on Nutrient Chemical Composition. Significant gene transformation effects (P < 0.05) were observed in protein content (CP, TP), carbohydrate components (sugars, starch, ADF, and NFC), and NPN but not for ash, EE, or ADL (Table 2). Double transgene alfalfa as a group (Lc1C1 and Lc3C1) showed significantly higher CP content (P < 0.05) than single transgene alfalfa (C1, Lc1, and Lc3) but was no different from NT alfalfa. The NPN content was approximately 3 to 4% in most tested populations, except for C1 and Lc1C1 populations, which had significantly higher NPN (P < 0.05). This indicated that C1 significantly increased the NPN content whether it was expressed alone or with Lc1, but such an impact was hindered by the Lc3 expression. Because of higher NPN content, the TP content in Lc1C1 was significantly lower (P < 0.05) than that in Lc3C1. The double transgene alfalfa (Lc1C1 and Lc3C1) had a similar but significantly lower sugar content than either NT (P < 0.01) or single gene alfalfas (P < 0.01), indicating that the combination of Lc and C1 transgenes has a synergic reducing effect on the alfalfa sugar content. A single expression of C1 had a higher starch content (comparable to NT) than all Lc expressed alfalfa, indicating a negative effect of Lc expression on starch content. The starch content in all transformed alfalfa and their parent NT genotype was significantly higher than that in ACG possibly because of the breeding clone background. ACG alfalfa had the lowest NFC content, followed by double transgene alfalfa populations with single
gene transformed alfalfa populations were positive for monomeric and polymeric proanthocyanidins, while C1transgenic, NT, or ACG alfalfa did not accumulate either type of proanthocyanidin (Table 1). The monomeric Table 1. Proanthocyanidin Content of Lc-bHLH (Lc) and C1-MYB (C1) Single Gene and Double Gene Transformed Alfalfa Compared to Their Nontransgenic Parent Genotype NT and AC Grazeland Variety (Monomeric and Polymeric Proanthocyanidins) alfalfa population
monomeric flavan-3-ol contents
C1 LC1 LC3 Lc1C1 Lc3C1 NT ACG SEM P value
nd 212.1 222.8 450.1 311.4 nd nd 90.61 0.430
Lc vs LcC1
0.08
polymeric proanthocyanidins
total
μg/g of DM nd 250.1 352.5 323.3 171.4 nd nd 78.95 0.486 Contrast P Value 0.50
nd 462.2 575.3 773.4 482.8 nd nd 136.75 0.474 0.43
proanthocyanidin content in LcC1 double gene alfalfa ranged from 311.4 to 450.1 μg/g of DM, while it tended to be higher (P = 0.08) than in Lc single gene alfalfa. The polymeric proanthocyanidin content was similar (mean 274 μg/g of DM; P > 0.05) among all of the alfalfa carrying Lc genes, with no significant differences detected between Lc single and LcC1 double transgenic groups. Jonker et al.11 reported that the accumulation of proanthocyanidins reduced the foaming formation and stability of alfalfa. The average total proanthocyanidin content in LcC1 double gene alfalfa was approximately 617 μg/g of DM, which was numerically higher than that of Lc single gene alfalfa (473 μg/g of DM).
Table 2. Nutrient Chemical Composition of Lc-bHLH (Lc) and C1-MYB (C1) Single Gene and Double Gene Transformed Alfalfa Compared to Their Nontransgenic Parent Genotype NT and Cultivar AC Grazelanda alfalfa population
ASH
CP
NPN
TP1
C1 LC1 LC3 Lc1C1 Lc3C1 NT ACG SEM P value
7.4 7.6 6.7 7.2 7.2 6.8 7.7 0.36 0.53
19.7ab 18.1b 19.5ab 20.7ab 21.2ab 20.1ab 22.3a 0.73 0.04
7.8a 2.9b 3.8b 8.3a 3.9b 4.5b 3.3b 0.33
