María Lorena Falcone Ferreyra

Flavonoids: biosynthesis, biological functions, and biotechnological applications

Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.

Cloning and characterization of a UV-B-inducible maize flavonol synthase

Flavonols are important compounds for conditional male fertility in maize (Zea mays) and other crops, and they also contribute to protecting plants from UV-B radiation. However, little continues to be known on how maize and other grasses synthesize flavonols, and how flavonol biosynthesis is regulated. By homology with an Arabidopsis flavonol synthase (AtFLS1), we cloned a maize gene encoding a protein (ZmFLS1) capable of converting the dihydrokaempferol (DHK) and dihydroquercetin (DHQ) dihydroflavonols to the corresponding flavonols, kaempferol (K) and quercetin (Q). Moreover, ZmFLS1 partially complements the flavonol deficiency of the Arabidopsis fls1 mutant, and restores anthocyanin accumulation to normal levels. We demonstrate that ZmFLS1 is under the control of the anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. Indeed, using chromatin immunoprecipitation (ChIP) experiments, we establish that ZmFLS1 is an immediate direct target of the P1 and C1/R regulatory complexes, revealing similar control as for earlier steps in the maize flavonoid pathway. Highlighting the importance of flavonols in UV-B protection, we also show that ZmFLS1 is induced in maize seedlings by UV-B, and that this induction is in part mediated by the increased expression of the P1, B and PL1 regulators. Together, our results identify a key flavonoid biosynthetic enzyme so far missed in maize and other monocots, and illustrate mechanisms by which flavonol accumulation is controlled in maize.

Plant L10 Ribosomal Proteins Have Different Roles during Development and Translation under Ultraviolet-B Stress

Ribosomal protein L10 (RPL10) proteins are ubiquitous in the plant kingdom. Arabidopsis (Arabidopsis thaliana) has three RPL10 genes encoding RPL10A to RPL10C proteins, while two genes are present in the maize (Zea mays) genome (rpl10-1 and rpl10-2). Maize and Arabidopsis RPL10s are tissue-specific and developmentally regulated, showing high levels of expression in tissues with active cell division. Coimmunoprecipitation experiments indicate that RPL10s in Arabidopsis associate with translation proteins, demonstrating that it is a component of the 80S ribosome. Previously, ultraviolet-B (UV-B) exposure was shown to increase the expression of a number of maize ribosomal protein genes, including rpl10. In this work, we demonstrate that maize rpl10 genes are induced by UV-B while Arabidopsis RPL10s are differentially regulated by this radiation: RPL10A is not UV-B regulated, RPL10B is down-regulated, while RPL10C is up-regulated by UV-B in all organs studied. Characterization of Arabidopsis T-DNA insertional mutants indicates that RPL10 genes are not functionally equivalent. rpl10A and rpl10B mutant plants show different phenotypes: knockout rpl10A mutants are lethal, rpl10A heterozygous plants are deficient in translation under UV-B conditions, and knockdown homozygous rpl10B mutants show abnormal growth. Based on the results described here, RPL10 genes are not redundant and participate in development and translation under UV-B stress.

Flavonols Protect Arabidopsis Plants against UV-B Deleterious Effects

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New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

Arabidopsis RIBOSOMAL PROTEIN L10 family members are nonredundant and contribute differentially to male gametophyte functionality and UV-B stress responses. The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing ProRPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.

The identification of maize and arabidopsis type I FLAVONE SYNTHASEs links flavones with hormones and biotic interactions

Two novel flavone synthase enzymes from maize and Arabidopsis integrate flavone metabolism with hormone and biotic stress responses. Flavones are a major group of flavonoids with diverse functions and are extensively distributed in land plants. There are two different classes of FLAVONE SYNTHASE (FNS) enzymes that catalyze the conversion of the flavanones into flavones. The FNSI class comprises soluble Fe2+/2-oxoglutarate-dependent dioxygenases, and FNSII enzymes are oxygen- and NADPH-dependent cytochrome P450 membrane-bound monooxygenases. Here, we describe the identification and characterization of FNSI enzymes from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana). In maize, ZmFNSI-1 is expressed at significantly higher levels in silks and pericarps expressing the 3-deoxy flavonoid R2R3-MYB regulator P1, suggesting that ZmFNSI-1 could be the main enzyme for the synthesis of flavone O-glycosides. We also show here that DOWNY MILDEW RESISTANT6 (AtDMR6), the Arabidopsis homologous enzyme to ZmFNSI-1, has FNSI activity. While dmr6 mutants show loss of susceptibility to Pseudomonas syringae, transgenic dmr6 plants expressing ZmFNSI-1 show similar susceptibility to wild-type plants, demonstrating that ZmFNSI-1 can complement the mutant phenotype. AtDMR6 expression analysis showed a tissue- and developmental stage-dependent pattern, with high expression in cauline and senescing leaves. Finally, we show that Arabidopsis cauline and senescing leaves accumulate apigenin, demonstrating that Arabidopsis plants have an FNSI activity involved in the biosynthesis of flavones. The results presented here also suggest cross talk between the flavone and salicylic acid pathways in Arabidopsis; in this way, pathogens would induce flavones to decrease salicylic acid and, hence, increase susceptibility.

Purification and physical and kinetic characterization of a photosynthetic NADP-dependent malic enzyme from the CAM plant Aptenia cordifolia

Two isoforms of NADP-dependent malic enzyme (NADP-ME) with the same molecular mass 72 kDa and different isoelectric points, 6.1 and 6.4, were found in crude extracts from the leaves of Aptenia cordifolia , a constitutive CAM plant. In the roots, only one isoform of 72 kDa was found, with a pI of 6.1. The isoform of pI 6.4 was partially purified from leaves to a final specific activity of 30.14 U mg � 1 ,a value similar to the photosynthetic isozymes. This enzyme showed a native mass of 264 kDa, suggesting a homotetramer. An optimal pH of 7.3 and Km values for NADP and L-malate 13 mM and 1.1 mM, respectively, were determined. The enzymatic activities and the level of immunoreactive protein did not vary with the day/night cycle. The enzyme was strongly and competitively inhibited by oxaloacetate (OAA), L-aspartate and phosphoenolpyruvate (PEP) and to a lesser degree by citrate, suggesting that NADP-ME activity might be subject to metabolite control. At night, high levels of OAA, L-aspartate and citrate might inhibit NADP-ME, avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of L-malate. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Evolution and Expression of Tandem Duplicated Maize Flavonol Synthase Genes

Flavonoids are specialized compounds widely distributed and with diverse functions throughout the plant kingdom and with several benefits for human health. In particular, flavonols, synthesized by flavonol synthase (FLS), protect plants against UV-B radiation and are essential for male fertility in maize and other plants. We have recently characterized a UV-B inducible ZmFLS1, corresponding to the first to be described in monocot plants. Interestingly, the new assembly of the B73 maize genome revealed the presence of a second putative FLS gene (ZmFLS2), with very high identity with ZmFLS1. ZmFLSs expression was analyzed in different maize tissues, and by combining electrophoretic mobility shift assays and transient expression experiments, we show that both genes are direct targets of anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. ZmFLS expression analyses show higher levels of both transcripts in high altitude landraces than inbred lines, and both genes are regulated by UV-B radiation in all lines analyzed. Moreover, the high sequence conservation of the ZmFLS promoters between maize lines suggests that the differences observed in ZmFLS expression are due to allelic variations in the transcription factors that regulate their activities. Finally, we generated pFLS1::FLS1-RFP transgenic plants and analyzed ZmFLS1 expression in different maize tissues; we found that this enzyme is localized in the ER and the perinuclear region.

Arabidopsis L10 ribosomal proteins in UV-B responses

Ribosomal protein L10 (RPL10) is an ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; however, increasing evidences indicate that RPL10 from various organisms has multiple extra ribosomal functions, besides being a constituent of ribosome and its role in translation. Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C. Previously, we found that in maize and in A. thaliana, UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B. Here, we show that RPL10s are differentially regulated by UV-B in a dosage and time dependent manner: RPL10C is induced, RPL10B is down regulated at high UV-B intensity, and RPL10A is not UV-B regulated. In addition, by coimmunoprecipitation studies using RPL10 antibodies and proteins from control and UV-B irradiated Arabidopsis plants, we demonstrate that RPL10 associates with different proteins under the two different conditions, including nuclear proteins, suggesting that at least one isoform may have extra-ribosomal roles.

A role for β,β-xanthophylls in Arabidopsis UV-B photoprotection

Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate β,β-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from β,β-xanthophylls). Plants accumulating β,β-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that β,β-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B.