Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc    

Abstract

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5Ј-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5Ј-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (ϩ)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.The ability to manipulate flavonoid biosynthesis in crop plants is gaining rapidly in importance as new economically important uses are found in the areas of nutraceuticals, food quality, and feed quality. The introduction of proanthocyanidin (PA, a flavonoid polymer) into alfalfa (Medicago sativa) forage is particularly important to ruminant livestock producers. Proanthocyanidins eliminate pasture bloat, improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass), reduce greenhouse gases, reduce gastrointestinal parasites, and inhibit insect feeding (Waghorn, 1990; Neizen et al., 1995 Neizen et al., , 1998 Aerts et al., 1999; Muir et al., 1999; McMahon et al., 2000). Alfalfa forage (leaf and stem tissues) accumulate anthocyanins only at senescence or locally under some stress conditions such as insect feeding (Goplen et al., 1980). No known conditions induce proanthocyanidins in alfalfa forage, although they are structurally related to anthocyanins. However, these compounds do accumulate in seed coats (Koupai-Abyazani et al., 1993).Anthocyanins and proanthocyanidins share early and middle steps of the flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase, flavan...

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

“…So far, it has not been fully successful, although intensive efforts have been made via the introduction of positive regulators of the PA pathway into alfalfa (Medicago sativa) or white clover; these genes include the transcription factors Lc (Ray et al, 2003), LAP1 ), MtWD40-1 , and MtPAR (Verdier et al, 2012). Although increased levels of anthocyanins and/or DMACA-reactive PA-like compounds could be detected by HPLC/UPLC, the level of PAs was far lower than desired, and the endogenous ANR genes were not greatly up-regulated Peel et al, 2009;Verdier et al, 2012).…”

A WD40 Repeat Protein fromMedicago truncatulaIs Necessary for Tissue-Specific Anthocyanin and Proanthocyanidin Biosynthesis But Not for Trichome Development

“…Successful examples of engineering anthocyanin production include ectopic expression of the Myb transcription factors Production of Anthocyanin Pigment1 (PAP1) in tobacco (Nicotiana tabacum) and Arabidopsis (Borevitz et al, 2000;Xie et al, 2006), Legume Anthocyanin Production1 in alfalfa and white clover (Trifolium repens; Peel et al, 2009), and the maize bHLH transcriptional regulators Lc and Sn in alfalfa and Lotus corniculatus, respectively (Ray et al, 2003;Robbins et al, 2003). Coexpression of PAP1 and TT2 led to the accumulation of detectable PA levels in Arabidopsis, although the plants did not survive .…”

In vitro ruminal digestion of anthocyanidin-containing alfalfa transformed with the maize Lc regulatory gene

“…Lc1, Lc2, Lc3 and Lc4 are genotypes developed from an anthocyanidin and proanthocyanidin-free eastern Canadian breeding genotype N4-2-2 developed at the University of Guelph (McKersie et al 1993;Ray et al 2003). The Lc genotypes were developed as interim germplasm in a longer-term study undertaken to produce "Tannin Alfalfa" with moderately high amounts of proanthocyanidins and higher forage quality.…”

“…The filtrates were adjusted to 50 mL using the same methanol-HCl solution, and absorbance values were measured at 530 nm (Spectronic 1001 Plus, Milton Roy, New York, NY). Background absorbance not due to red anthocyanidins was measured in the NT alfalfa extract and subtracted from absorbance for Lc-transgenic alfalfa extracts according to Ray et al (2003). Concentration curves were developed using cyanidin chloride (Alexis Biochemicals, San Diego, CA), and the concentration of anthocyanidin in the alfalfa genotypes was expressed as cyanidin equivalents.…”