Data_Sheet_3_Features of Activity of the Phenylpropanoid Biosynthesis Pathway in Melanin-Accumulating Barley Grains.PDF

Barley (Hordeum vulgare L.) grain pigmentation is caused by two types of phenolic compounds: anthocyanins (which are flavonoids) give a blue or purple color, and melanins (which are products of enzymatic oxidation and polymerization of phenolic compounds) give a black or brown color. Genes Ant1 and Ant2 determine the synthesis of purple anthocyanins in the grain pericarp, whereas melanins are formed under the control of the Blp1 gene in hulls and pericarp tissues. Unlike anthocyanin synthesis, melanin synthesis is poorly understood. The objective of the current work was to reveal features of the phenylpropanoid biosynthesis pathway functioning in melanin-accumulating barley grains. For this purpose, comparative transcriptomic and metabolomic analyses of three barley near-isogenic lines accumulating anthocyanins, melanins, or both in the grain, were performed. A comparative analysis of mRNA libraries constructed for three stages of spike development (booting, late milk, and early dough) showed transcriptional activation of genes encoding enzymes of the general phenylpropanoid pathway in all the lines regardless of pigmentation; however, as the spike matured, unique transcriptomic patterns associated with melanin and anthocyanin synthesis stood out. Secondary activation of transcription of the genes encoding enzymes of the general phenylpropanoid pathway together with genes of monolignol synthesis was revealed in the line accumulating only melanin. This pattern differs from the one observed in the anthocyanin-accumulating lines, where — together with the genes of general phenylpropanoid and monolignol synthesis pathways — flavonoid biosynthesis genes were found to be upregulated, with earlier activation of these genes in the line accumulating both types of pigments. These transcriptomic shifts may underlie the observed differences in concentrations of phenylpropanoid metabolites analyzed in the grain at a late developmental stage by high-performance liquid chromatography. Both melanin-accumulating lines showed an increased total level of benzoic acids. By contrast, anthocyanin-accumulating lines showed higher concentrations of flavonoids and p-coumaric and ferulic acids. A possible negative effect of melanogenesis on the total flavonoid content and a positive influence on the anthocyanin content were noted in the line accumulating both types of pigments. As a conclusion, redirection of metabolic fluxes in the phenylpropanoid biosynthesis pathway occurs when melanin is synthesized.

大麦（Hordeum vulgare L.）籽粒着色由两类酚类化合物介导：花青素（anthocyanins，属于类黄酮）赋予籽粒蓝或紫色，而黑色素（melanins，由酚类化合物经酶促氧化聚合生成）则赋予籽粒黑或褐色。Ant1与Ant2基因调控籽粒果皮中紫色花青素的合成，而黑色素则在颖壳与果皮组织中受Blp1基因调控合成。相较于花青素合成途径，目前对黑色素合成的分子机制认知仍较为匮乏。本研究旨在解析黑色素积累型大麦籽粒中苯丙烷类生物合成途径的功能特征。为此，本研究针对三类分别积累花青素、黑色素或同时积累两类色素的大麦近等基因系，开展了比较转录组学与代谢组学分析。对构建于穗发育三个阶段（孕穗期、乳熟后期、蜡熟初期）的mRNA文库进行比较分析后发现，无论籽粒色素类型如何，所有品系中编码苯丙烷类通用代谢途径酶类的基因均出现转录激活；但随着穗部成熟，与黑色素及花青素合成相关的独特转录组模式逐渐显现。仅积累黑色素的品系中，观测到苯丙烷类通用代谢途径与单木质醇合成途径相关基因的二次转录激活。该模式与花青素积累型品系存在显著差异：在花青素积累型品系中，除苯丙烷类通用代谢与单木质醇合成途径基因外，类黄酮生物合成基因亦呈现上调表达，且在同时积累两类色素的品系中，此类基因的激活时间更早。上述转录组变化可解释本研究通过高效液相色谱法（high-performance liquid chromatography）在籽粒发育后期检测到的苯丙烷类代谢物浓度差异。两类黑色素积累型品系的苯甲酸类总含量均显著升高；而花青素积累型品系的类黄酮、对香豆酸与阿魏酸浓度则更高。在同时积累两类色素的品系中，观测到黑色素生成可能对总类黄酮含量存在负调控效应，而对花青素含量具有正调控作用。综上，黑色素合成过程中，苯丙烷类生物合成途径的代谢流发生了重定向。