FUNCTIONAL CHARACTERIZATION AND EXPRESSION ANALYSIS OFp-HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM THE GREEN ALGACHLAMYDOMONAS REINHARDTII(CHLOROPHYTA)

Abstract

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) is very important in prenylquinone biosynthesis in all photosynthetic organisms. In this study, we present the functional characterization and expression analysis of HPPD from the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. Recombinant HPPD1 enzyme was purified and characterized. Kinetic analysis revealed a K m of 49 lM for p-hydroxyphenylpyruvate, similar to other HPPDs. The size of HPPD subunit was estimated as 47 kDa by SDS-PAGE, in accordance with the predicted molecular size after HPPD1 cDNA sequence. However, native HPPD1 enzyme showed an apparent molecular mass of 188 kDa and a homotetrameric structure, which suggests a reconsideration of the idea that all eukaryotic HPPDs have a homodimeric structure while all prokaryotic HPPDs are homotetramers. Expression analysis by Northern blot revealed that hppd1 expression is strongly up-regulated by low temperature and poorly regulated by high temperature, darkness, or moderate light changes, suggesting that Chlamydomonas HPPD may play an important role in the synthesis of tocopherols and ⁄ or plastoquinones under stress conditions in the physiological context of the adaptation to growth at low temperatures.

γ‐Tocopherol methyltransferase from the green alga Chlamydomonas reinhardtii: functional characterization and expression analysis

“…Other regulatory elements related to light (SORLIP1AT, SORLIP2AT) were identified at −670, −625, −355, −352, −280, −276. As it was previously shown for Chlamydomonas hppd1 gene (Gálvez‐Valdivieso et al 2010), we have found a GCCGCC domain (–733) in the promoter of tmt1 that seems to be involved in regulation by jasmonic acid (Brown et al 2003), a molecule that plays a role in signaling stress processes where free radicals of unsaturated fatty acids and ROS are generated. There seems to be a biochemical and metabolic basis for a regulatory cross‐talk between tocopherols and jasmonate biosynthesis.…”

“…In the photosynthetic bacteria Synechocystis , tocopherols can protect membranes from lipid peroxidation caused by an exposure to exogenous polyunsaturated fatty acids in high light conditions (Maeda et al 2005). In a previous report (Gálvez‐Valdivieso et al 2010) we showed that Chlamydomonas hppd1 , a gene coding for the enzyme that controls the initial step of tocopherols and plastocyanins biosynthesis, is upregulated by low temperature, which suggested an induction of tocopherol synthesis under these circumstances. This hypothesis is consistent with results obtained by Bergmuller et al (2003) who, after exposing Arabidopsis leaves to 4°C for 5 days, observed that levels of γ ‐tocopherol increased more than 10 times, whereas α ‐tocopherol rose only 1.5 times.…”

“…hppd1 and tmt1 also seem to be differently regulated by light. While hppd1 was downregulated by high light (Gálvez‐Valdivieso et al 2010), tmt1 seems to be strongly upregulated. This differential regulation of hppd1 and tmt1 supports that γ ‐tocopherol might have a particular role in the adaptation of Chlamydomonas to low temperature.…”

Combined effect of temperature and bleaching herbicides on photosynthesis, pigment and fatty acid composition ofChlamydomonas reinhardtii

“…It is also known that Nf interacts with the cofactors NADP or plastoquinone for the phytoene desaturase enzyme (Breitenbach et al, 2001). Low temperature has been associated with a significant increase in plastoquinone (Griffith et al, 1984;Gálvez-Valdivieso et al, 2010). It is therefore possible that at low temperature the effect of Nf is less important since there is a competition with the larger amount of plastoquinone available.…”

Morphological and Molecular Characterization of Three Endolichenic Isolates of Xylaria (Xylariaceae), from Cladonia curta Ahti & Marcelli (Cladoniaceae)

“…berteri , the group of taxa reported as endophytic fungi of angiosperms and the species isolated in this work, which are recorded as endolichenic for the first time, could be a species complex. Considering that the characterization of the anamorph of Xylaria is required to separate closely related taxa in certain species complexes [52,53], this work provides basic information regarding characterization of the anamorph.…”