Sebastián P. Rius

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.

Characterization of Arabidopsis Lines Deficient in GAPC-1, a Cytosolic NAD-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase

Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants.

Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase

Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.

Effect of mitochondrial dysfunction on carbon metabolism and gene expression in flower tissues of Arabidopsis thaliana.

We characterized the transcriptomic response of transgenic plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase subunit 9. The u-ATP9 transgene driven by A9 and APETALA3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and adenine nucleotides (ATP, ADP) levels without changes in the ATP/ADP ratio. Furthermore, we measured an increase in ROS accumulation and a decrease in glutathione and ascorbate levels with a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by qRT-PCR and enzymatic or functional tests, showed dramatic changes in u-ATP9 plants. Both lines display a modification in the expression of various genes involved in carbon, lipid, and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial respiration, protein synthesis, and degradation are affected. Moreover, the levels of several mRNAs encoding for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone responses, suggesting that several signaling pathways overlap. Indeed, the transcriptome data revealed that the mitochondrial dysfunction dramatically alters the expression of genes involved in signaling pathways, including those related to ethylene, absicic acid, and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this report may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.

Analysis of the P1 promoter in response to UV-B radiation in allelic variants of high-altitude maize

BackgroundPlants living at high altitudes are typically exposed to elevated UV-B radiation, and harbor mechanisms to prevent the induced damage, such as the accumulation of UV-absorbing compounds. The maize R2R3-MYB transcription factor P1 controls the accumulation of several UV-B absorbing phenolics by activating a subset of flavonoid biosynthetic genes in leaves of maize landraces adapted to high altitudes.ResultsHere, we studied the UV-B regulation of P1 in maize leaves of high altitude landraces, and we investigated how UV-B regulates P1binding to the CHS promoter in both low and high altitude lines. In addition, we analyzed whether the expansion in the P1 expression domain between these maize landraces and inbred lines is associated to changes in the molecular structure of the proximal promoter, distal enhancer and first intron of P1. Finally, using transient expression experiments in protoplasts from various maize genotypes, we investigated whether the different expression patterns of P1 in the high altitude landraces could be attributed to trans- or cis-acting elements.ConclusionsTogether, our results demonstrate that, although differences in cis-acting elements exist between the different lines under study, the different patterns of P1 expression are largely a consequence of effects in trans.

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana.

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus cells and the pRSETB vector was successful for expression of a soluble His(6)-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.

P1 Epigenetic Regulation in Leaves of High Altitude Maize Landraces: Effect of UV-B Radiation

P1 is a R2R3-MYB transcription factor that regulates the accumulation of a specific group of flavonoids in maize floral tissues, such as flavones and phlobaphenes. P1 is also highly expressed in leaves of maize landraces adapted to high altitudes and higher levels of UV-B radiation. In this work, we analyzed the epigenetic regulation of the P1 gene by UV-B in leaves of different maize landraces. Our results demonstrate that DNA methylation in the P1 proximal promoter, intron1 and intron2 is decreased by UV-B in all lines analyzed; however, the basal DNA methylation levels are lower in the landraces than in B73, a low altitude inbred line. DNA demethylation by UV-B is accompanied by a decrease in H3 methylation at Lys 9 and 27, and by an increase in H3 acetylation. smRNAs complementary to specific regions of the proximal promoter and of intron 2 3′ end are also decreased by UV-B; interestingly, P1 smRNA levels are lower in the landraces than in B73 both under control conditions and after UV-B exposure, suggesting that smRNAs regulate P1 expression by UV-B in maize leaves. Finally, we investigated if different P1 targets in flower tissues are also regulated by this transcription factor in response to UV-B. Some targets analyzed show an induction in maize landraces in response to UV-B, with higher basal expression levels in the landraces than in B73; however, not all the transcripts analyzed were found to be regulated by UV-B in leaves.

Genome walking by Klenow polymerase.

Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species.

HAC1 and HAF1 Histone Acetyltransferases Have Different Roles in UV-B Responses in Arabidopsis

Arabidopsis has 12 histone acetyltransferases grouped in four families: the GNAT/HAG, the MYST/HAM, the p300/CBP/HAC and the TAFII250/HAF families. We previously showed that ham1 and ham2 mutants accumulated higher damaged DNA after UV-B exposure than WT plants. In contrast, hag3 RNA interference transgenic plants showed less DNA damage and lower inhibition of plant growth by UV-B, and increased levels of UV-B-absorbing compounds. These results demonstrated that HAM1, HAM2, and HAG3 participate in UV-B-induced DNA damage repair and signaling. In this work, to further explore the role of histone acetylation in UV-B responses, a putative function of other acetyltransferases of the HAC and the HAF families was analyzed. Neither HAC nor HAF acetyltrasferases participate in DNA damage and repair after UV-B radiation in Arabidopsis. Despite this, haf1 mutants presented lower inhibition of leaf and root growth by UV-B, with altered expression of E2F transcription factors. On the other hand, hac1 plants showed a delay in flowering time after UV-B exposure and changes in FLC and SOC1 expression patterns. Our data indicate that HAC1 and HAF1 have crucial roles for in UV-B signaling, confirming that, directly or indirectly, both enzymes also have a role in UV-B responses.