Freixas Coutin José A, Munholland Seth, Silva Anjali, Subedi Sanjeena, Lukens Lewis, Crosby William L, Pauls K Peter, Bozzo Gale G
Department of Plant Agriculture, University of Guelph, 50 Stone Rd E., Guelph, ON, N1G 2W1, Canada.
Department of Biological Sciences, University of Windsor, 401 Sunset Ave, Windsor, ON, N9B 3P4, Canada.
BMC Plant Biol. 2017 May 25;17(1):89. doi: 10.1186/s12870-017-1037-z.
Edible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown.
Seed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products.
Proanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.
在收获后储存期间会变黑的可食用干豆（菜豆），其等级低于未变黑的同类产品，市场销路也较差。易感基因型种子的种皮变黑取决于原花青素的可用性及其随后氧化为活性醌。缺乏这种收获后变黑特性的成熟蔓越莓豆往往缺乏原花青素，尽管这种代谢现象的潜在分子和生化决定因素尚不清楚。
在一个易变黑的蔓越莓豆重组自交系（RIL）中，种皮原花青素水平随着植株成熟而增加，而在不易变黑的RIL植株种子中不存在这些代谢物。RNA测序（RNA-seq）分析用于监测种皮转录组随菜豆发育的变化，转录水平以每百万映射片段中外显子每千碱基的片段数来衡量。在易变黑和不易变黑的蔓越莓豆RIL之间共有1336个基因差异表达。原花青素生物合成途径的结构和调控基因在易变黑的RIL种皮中上调。主成分分析确定，两个功能未知基因和三个原花青素生物合成基因（黄酮醇3-羟化酶1、二氢黄酮醇4-还原酶1和花青素还原酶1（PvANR1））的转录水平变化与易变黑的蔓越莓豆RIL种皮中原花青素积累高度相关。HPLC-DAD分析表明，重组PvANR1的体外活性依赖于NADPH，含有花青素的测定产生表儿茶素和儿茶素；高花青素底物水平抑制了这两种产物的形成。
原花青素氧化是双子叶种子收获后种皮变黑的先决条件。在模式植物物种中，原花青素的积累依赖于生物合成基因的上调。在本研究中，蔓越莓豆种皮中原花青素的产生与种子成熟期间PvANR1转录本的增加密切相关。在NADPH存在的情况下，PvANR1将生理相关底物花青素转化为表儿茶素和儿茶素。
