RESUMEN
Polyphenol oxidase (PPO) is an oxidoreductase. In damaged plant tissues, it catalyzes enzymatic browning by oxidizing o-diphenols to highly reactive o-quinones, which polymerize producing heterogeneous dark polymer melanin. In intact tissues, functions of PPO are not well understood. The aim of the study was to investigate the barley PPO gene family and to reveal the possible involvement of Ppo genes in melanization of barley grain, which is controlled by the Blp1 gene. Based on known barley Ppo genes on chromosome 2H (Ppo1 and Ppo2), two additional genes-Ppo3 and Ppo4-were found on chromosomes 3H and 4H, respectively. These genes have one and two exons, respectively, contain a conserved tyrosinase domain and are thought to be functional. Comparative transcriptional analyzes of the genes in samples of developing grains (combined hulls and pericarp tissues) were conducted in two barley lines differing by melanin pigmentation. The genes were found to be transcribed with increasing intensity (while grains mature) independently from the grain color, except for Ppo2, which is transcribed only in black-grained line i:BwBlp1 accumulating melanin in grains. Analysis of this gene's expression in detached hulls and pericarps showed its elevated transcription in both tissues in comparison with yellow ones, while it was significantly higher in hulls than in pericarp. Segregation analysis in two F2 populations obtained based on barley genotypes carrying dominant Blp1 and recessive ppo1 (I) and dominant Blp1 and recessive ppo1 and ppo2 (II) was carried out. In population I, only two phenotypic classes corresponding to parental black and white ones were observed; the segregation ratio was 3 black to 1 white, corresponding to monogenic. In population II, aside from descendants with black and white grains, hybrids with a gray phenotype - light hulls and dark pericarp - were observed; the segregation ratio was 9 black to 3 gray to 4 white, corresponding to the epistatic interaction of two genes. Most hybrids with the gray phenotype carry dominant Blp1 and a homozygous recessive allele of Ppo2. Based on transcription and segregation assays one may conclude involvement of Ppo2 but not Ppo1 in melanin formation in barley hulls.
RESUMEN
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.
RESUMEN
The word "melanin" refers to a group of high molecular weight, black, and brown pigments formed through the oxidation and polymerization of phenolic compounds. This pigment is present in all kingdoms of living organisms, but it remains the most enigmatic pigment in plants. The poor solubility of melanin in particular solvents and its complex polymeric nature significantly constrain its study. Plant melanin synthesis is mostly associated with the enzymatic browning reaction that occurs in wounded plant tissues. This reaction occurs when, due to the disruption of cellular compartmentation, the chloroplast-located polyphenol oxidases (PPOs) release from the chloroplast and interact with their vacuolar substrates to produce o-quinones, which in turn polymerize to melanin. Furthermore, the presence of melanin in intact seed tissues has been demonstrated by diagnostic physicochemical tests. Unlike the well-studied enzymatic browning reaction, little is known about how melanin is formed in seeds. Recent data have shown that it is a tightly controlled genetic process that involves many genes, among which the genes encoding PPOs might be key. The present article aims to provide an overview of the current knowledge on melanin in plants and to discuss future perspectives on its study in light of recent findings.
