Margaret Y. Gruber

New perspectives on proanthocyanidin biochemistry and molecular regulation

Our understanding of proanthocyanidin (syn. condensed tannin) synthesis has been recently extended by substantial developments concerning both structural and regulatory genes. A gene encoding leucoanthocyanidin reductase has been obtained from the tropical forage, Desmodium uncinatum, with the latter enzyme catalyzing formation of (+)-catechin. The BANYULS gene in Arabidopsis thaliana, previously proposed to encode leucoanthocyanidin reductase or to regulate proanthocyanidin biosynthesis, has been shown instead to encode anthocyanidin reductase, which in turn converts anthocyanidins (pelargonidin, cyanidin, or delphinidin) into 2,3-cis-2R,3R-flavan-3-ols (respectively, (-)-epiafzelechin, (-)-epicatechin and (-)-epigallocatechin). However, the enzyme which catalyzes the polymerization reaction remains unknown. Nevertheless, a vacuolar transmembrane protein TT12, defined by the Arabidopsis tt12 mutant, is involved in transport of proanthocyanidin polymer into the vacuole for accumulation. Six different types of regulatory elements, e.g. TFIIIA-like, WD-40-like, WRKY-like, MADS-box-like, myb-like, and bHLH (myc-like), have been cloned and identified using mutants from Arabidopsis (tt1, ttg1, ttg2, tt2, tt16, tt2, tt8) and two other species (Hordeum vulgare [ant13] and Lotus spp [tan1]). Accordingly, increases in proanthocyanidin levels have been induced in the the worlds major forage, alfalfa. These advances may now lead to a detailed understanding of how PA synthesis is controlled and to useful alterations in proanthocyanidin concentration for the improvement of forage species, pulses, and other crop plants.

Expression of anthocyanins and proanthocyanidins after transformation of alfalfa with maize Lc.

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 5′-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 5′-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 (+)-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.

An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches

BackgroundFunctional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenised plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.ResultsWe have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T3 generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.ConclusionThis publicly available population provides an additional tool for plant researchers to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.

Purple Canola: Arabidopsis PAP1 Increases Antioxidants and Phenolics in Brassica napus Leaves

Anthocyanins, other flavonoids, and phenolic acids belong to a group of plant natural products with antioxidant activity and may play important roles in plant protection against biotic and abiotic stress and in protection against human diseases. In the present study, the Arabidopsis regulatory gene Production of Anthocyanin Pigment 1 (AtPAP1) was expressed in Brassica napus (canola), and its presence enhanced the antioxidant capacity in transgenic leaves up to 4-fold. Transgenic plants had intense purple coloration, cyanidin and pelargonidin levels were enhanced 50-fold, and quercetin and sinapic acid were 5-fold higher. Consistent with these phytochemical and biological changes, expression for most genes in the flavonoid and phenolic acid biosynthetic pathways was also stimulated.

Molecular basis of protein structure in proanthocyanidin and anthocyanin-enhanced Lc-transgenic alfalfa in relation to nutritive value using synchrotron-radiation FTIR microspectroscopy: A novel approach

To date there has been very little application of synchrotron radiation-based Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular structures in plant forage in relation to livestock digestive behavior and nutrient availability. Protein inherent structure, among other factors such as protein matrix, affects nutritive quality, fermentation and degradation behavior in both humans and animals. The relative percentage of protein secondary structure influences protein value. A high percentage of beta-sheets usually reduce the access of gastrointestinal digestive enzymes to the protein. Reduced accessibility results in poor digestibility and as a result, low protein value. The objective of this study was to use SRFTIRM to compare protein molecular structure of alfalfa plant tissues transformed with the maize Lc regulatory gene with non-transgenic alfalfa protein within cellular and subcellular dimensions and to quantify protein inherent structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling. Protein molecular structure revealed by this method included alpha-helices, beta-sheets and other structures such as beta-turns and random coils. Hierarchical cluster analysis and principal component analysis of the synchrotron data, as well as accurate spectral analysis based on curve fitting, showed that transgenic alfalfa contained a relatively lower (P<0.05) percentage of the model-fitted alpha-helices (29 vs. 34) and model-fitted beta-sheets (22 vs. 27) and a higher (P<0.05) percentage of other model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no difference (P>0.05) in the ratio of alpha-helices to beta-sheets (average: 1.4) and higher (P<0.05) ratios of alpha-helices to others (0.7 vs. 0.9) and beta-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The transgenic protein structures also exhibited no difference (P>0.05) in the vibrational intensity of protein amide I (average of 24) and amide II areas (average of 10) and their ratio (average of 2.4) compared with non-transgenic alfalfa. Cluster analysis and principal component analysis showed no significant differences between the two genotypes in the broad molecular fingerprint region, amides I and II regions, and the carbohydrate molecular region, indicating they are highly related to each other. The results suggest that transgenic Lc-alfalfa leaves contain similar proteins to non-transgenic alfalfa (because amide I and II intensities were identical), but a subtle difference in protein molecular structure after freeze drying. Further study is needed to understand the relationship between these structural profiles and biological features such as protein nutrient availability, protein bypass and digestive behavior of livestock fed with this type of forage.

Leaf flavonoids of the cruciferous species, Camelina sativa, Crambe spp., Thlaspi arvense and several other genera of the family Brassicaceae

Abstract The flavonoid profiles of 22 accessions of Camelina sativa and five other crucifer species, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus, and Sinapis alba , were studied by a combination of liquid, paper and thin layer chromatography. Flavonoids were confirmed by comparison of their characteristics, including colour under UV light, changes to colour under UV with fuming in NH 3 vapour, UV spectra and comparison of RF-values, with those of authentic standards. HPLC-mass spectroscopic data were obtained to confirm identities of several compounds. Flavonoids present in several other crucifer species were identified by TLC only. We report the accumulation of derivatives of the flavonols, quercetin, in C. sativa; quercetin and kaempferol in C. hispanica var. glabrata ; quercetin, kaempferol and isorhamnetin in B. napus ; and kaempferol and isorhamnetin in S. alba . Derivatives of the flavones, apigenin and luteolin, accumulate in C. abyssinica, C. hispanica var. hispanica and T. arvense leaves.

Enhanced seed carotenoid levels and branching in transgenic Brassica napus expressing the Arabidopsis miR156b gene.

The Arabidopsis AtmiR156b gene was expressed in Brassica napus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the seed-specific napin promoter. Seed carotenoid levels, branching habit, seed yield, and seed weight were examined in the transgenic B. napus. Our results demonstrated that constitutive expression of AtmiR156b in B. napus resulted in enhanced levels of seed lutein and beta-carotene and a 2-fold increase in the number of flowering shoots, whereas AtmiR156b driven by the napin promoter did not affect these traits. This suggested that enhancement of seed quality and shoot branching are both related to AtmiR156b expression patterns. Seed yield and seed weight varied significantly within the transgenic lines. However, one line was found to have enhanced seed carotenoid levels but unchanged seed weight or yield. These data suggest that AtmiR156b gene expression could be applied in plant breeding initiatives for enhancing carotenoid production in canola and other crop species.

Effect of flavonoids on feeding preference and development of the crucifer pest Mamestra configurata walker

Thirty-seven flavonoid compounds (9 flavones, 18 flavonols, 8 flavanones, and 2 flavanonols) were investigated for their effect on feeding choice with bertha armyworm (Mamestra configurata Walker; BAW). Feeding choice was dependent upon subtle differences in biochemical structure. Unsubstituted flavone and flavanone were the strongest feeding deterrents in the choice bioassay, while 7,4′-dihydroxyflavone and dihydroquercetin stimulated BAW to feed. The constitutive flavonoids of Brassica napus, isorhamnetin-3-sophoroside-7-glucoside and kaempferol-3,7-diglucoside, were effective deterrents when supplemented at concentrations higher than endogenous levels. In a no-choice bioassay, flavone reduced both larval weight as well as larval and pupal development time.

Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156 -controlled gene network

BackgroundThe Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known.ResultsIn this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15 and the proximal promoter sequence of miR156b.ConclusionsEnhanced miR156b expression in sk156 leads to the mutant phenotype including carotenoid levels in the seed through suppression of SPL15 and other SPL target genes. Moreover, SPL15 has a regulatory role not only for downstream components, but also for its own upstream regulator miR156b.

Pigmentation in the developing seed coat and seedling leaves of Brassica carinata is controlled at the dihydroflavonol reductase locus

Flavonoid differences between near-isogenic lines of yellow- and brown-seeded Brassica carinata were used to identify a genetic block in seed coat and seedling leaf pigment biosynthesis. Seed coat pigment in the brown-seeded line consisted of proanthocyanidins (condensed tannins), while anthocyanin was absent. Dihydroquercetin, dihydrokaempferol, quercetin and kaempferol accumulated only in the mature seed coat of the yellow-seeded line, indicating dihydroflavonol reductase (DFR) as an element of genetic control in pigment biosynthesis. DFR transcripts from the developing seed coat in the yellow-seeded line were absent or less abundant at 5-30 days after pollination compared to transcript levels in the brown-seeded line. Seedling leaves of the yellow-seeded line exhibited reduced expression of DFR and contained less anthocyanin compared to the respective tissues from plants of the brown-seeded line when grown at 25/20 degrees C (day/night). Cooler (18/15 degrees C) growing temperatures affected seedling leaf pigmentation, mature seed coat colouration and DFR expression in the yellow-seeded line. Comparable brown-seeded line tissues were unaffected by these temperature changes. These results are suggestive of a temperature-sensitive regulator of DFR in the yellow-seeded line of Brassica carinata which ultimately affects the formation of pigments in the seedling leaves and in the mature seed coats.