Paula Casati

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.

NADP‐malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways

NADP‐malic enzyme (NADP‐ME) is a widely distributed enzyme that catalyzes the oxidative decarboxylation of L‐malate. Photosynthetic NADP‐MEs are found in C4 bundle sheath chloroplasts and in the cytosol of CAM plants, while non‐photosynthetic NADP‐MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP‐MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP‐MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous enzyme.

Transcriptional and Metabolic Changes Associated to the Infection by Fusarium verticillioides in Maize Inbreds with Contrasting Ear Rot Resistance

Fusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.

DDM1 and ROS1 have a role in UV-B induced- and oxidative DNA damage in A. thaliana

Absorption of UV-B by DNA induces the formation of covalent bonds between adjacent pyrimidines. In maize and arabidopsis, plants deficient in chromatin remodeling show increased DNA damage compared to WT plants after a UV-B treatment. However, the role of enzymes that participate in DNA methylation in DNA repair after UV-B damage was not previously investigated. In this work, we analyzed how chromatin remodeling activities that have an effect on DNA methylation affects the repair of UV-B damaged DNA using plants deficient in the expression of DDM1 and ROS1. First, we analyzed their regulation by UV-B radiation in arabidopsis plants. Then, we demonstrated that ddm1 mutants accumulated more DNA damage after UV-B exposure compared to Col0 plants. Surprisingly, ros1 mutants show less CPDs and 6-4PPs than WT plants after the treatment under light conditions, while the repair under dark conditions is impaired. Transcripts for two photolyases are highly induced by UV-B in ros1 mutants, suggesting that the lower accumulation of photoproducts by UV-B is due to increased photorepair in these mutants. Finally, we demonstrate that oxidative DNA damage does not occur after UV-B exposure in arabidopsis plants; however, ros1 plants accumulate high levels of oxoproducts, while ddm1 mutants have less oxoproducts than Col0 plants, suggesting that both ROS1 and DDM1 have a role in the repair of oxidative DNA damage. Together, our data provide evidence that both DDM1 and ROS1, directly or indirectly, participate in UV-B induced- and oxidative DNA damage repair.

Recent Advances in Maize Nuclear Proteomic Studies Reveal Histone Modifications

The nucleus of eukaryotic organisms is highly dynamic and complex, containing different types of macromolecules including DNA, RNA, and a wide range of proteins. Novel proteomic applications have led to a better overall determination of nucleus protein content. Although nuclear plant proteomics is only at the initial phase, several studies have been reported and are summarized in this review using different plants species, such as Arabidopsis thaliana, rice, cowpea, onion, garden cress, and barrel clover. These include the description of the total nuclear or phospho-proteome (i.e., Arabidopsis, cowpea, onion), or the analysis of the differential nuclear proteome under different growth environments (i.e., Arabidopsis, rice, cowpea, onion, garden cress, and barrel clover). However, only few reports exist on the analysis of the maize nuclear proteome or its changes under various conditions. This review will present recent data on the study of the nuclear maize proteome, including the analysis of changes in posttranslational modifications in histone proteins.

Factors affecting the oligomeric state of NADP-malic enzyme from maize and wheat tissues: a chemical crosslinking study

The different aggregational states of maize and wheat NADP-malic enzyme as affected by pH, temperature and various metabolites have been studied by the combined use of intersubunit crosslinking and denaturing polyacrylamide gel electrophoresis. The association/dissociation equilibrium is a pH-dependent process: pH values above 8.0 promote the tetramer formation, while lowering the pH shifts the equilibria towards dimers and monomers. Below pH 6.0, most molecules exist as monomers. In the same way, the temperature governs the equilibria between the different oligomeric states. As the temperature is lowered from 42 to 0 degrees C, a progressive dissociation into dimers and monomers is observed. Excess enthalpies are negative in all cases, but the overall process demands an input of Gibbs free energy. Consequently, the protein dissociation is an entropy-driven process. The presence of Mg2+ or glycerol induces aggregation in both enzymes, while increasing the ionic strength produces the opposite effect. The results suggest that changes in the equilibria between monomer, dimer and tetramer of NADP-malic enzyme could be the molecular basis for an effective regulation of the enzyme activity in vivo.

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.

ZmMBD101 is a DNA binding protein that maintains Mutator elements chromatin in a repressive state in maize

In maize (Zea mays), as well as in other crops, transposable elements (TEs) constitute a great proportion of the genome. Chromatin modifications play a vital role in establishing transposon silencing and perpetuating the acquired repressive state. Nucleosomes associated with TEs are enriched for dimethylation of histone H3 at lysine 9 and 27 (H3K9me2 and H3K27me2, respectively), signals of repressive chromatin. Here, we describe a chromatin protein, ZmMBD101, involved in the regulation of Mutator (Mu) genes in maize. ZmMBD101 is localized to the nucleus and contains a methyl-CpG-binding domain (MBD) and a zinc finger CW (CW) domain. Transgenic lines with reduced levels of ZmMBD101 transcript present enhanced induction of Mu genes when plants are irradiated with UV-B. Chromatin immunoprecipitation analysis with H3K9me2 and H3K27me2 antibodies indicated that ZmMBD101 is required to maintain the levels of these histone repressive marks at Mu terminal inverted repeats (TIRs) under UV-B conditions. Although Mutator inactivity is associated with DNA methylation, cytosine methylation at Mu TIRs is not affected in ZmMBD101 deficient plants. Several plant proteins are predicted to share the simple CW-MBD domain architecture present in ZmMBD101. We hypothesize that plant CW-MBD proteins may also function to protect plant genomes from deleterious transposition.

NADP-Malic Enzyme from the C 3 -C 4 Intermediate Specie Flaveria Floridana

C4 plants have fully differentiated mesophyll (MC) and bundle sheath cells (BSC) which cooperate to fix CO2 by the C4 pathway. C4 plants are more efficient than C3 under some environmental conditions due to the higher CO2 concentration in BSC. C3-C4 intermediate species are thought to represent a stage in the evolutionary transition from the C3 to the C4 photosynthetic mechanism (1,2). MC of C3-C4 species function, as in C3 plants, fixing atmospheric CO2 through RuBisCO and generating C-2 compounds for the photosynthetic oxidation cycle. Two mechanisms are proposed to account for the low apparent photorespiration in these intermediate species. In one of them, which may be common to all intermediates, metabolites generated as a consequence of the RuBisCO oxygenase reaction in MC are metabolized in BSC and the CO2 released refixed by RuBisCO. In this way, reduced photorespiratory CO2 evolution occurs without the operation of a C4 cycle. In other class of intermediates, the operation of a limited C4 cycle between MC and BSC contributes to the further reduction of photorespiration. Flaveria floridana, a C3-C4 intermediate specie, was used in the present report to characterize different isoforms of NADP-malic enzyme (NADP-ME) and to evaluate their expression in different photosynthetic cell types. In previous studies, we detected three isoforms of the enzyme in various Flaveria intermediate species (3). One of them was found to be constitutively expressed in photosynthetic and non-photosynthetic tissues of the different species examined, while the other two isoforms were abundant only in photosynthetic tissues having partial or complete C4 photosynthesis. A correlation between these proteins, their function and the cDNAs already cloned (4) is necessary to understand the evolution of this protein among the different Flaveria species.