Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Abstract

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid ApH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.Light is essential for photosynthesis but potentially can damage the photosynthetic apparatus when the intensity exceeds photosynthetic capacity. Plants have evolved various mechanisms to cope with the excess-light conditions that, in fact, are experienced by most plants in natural environments. In higher plants, a highly conserved feedback mechanism, the zeaxanthin and antheraxanthin-dependent dissipation of energy in the light-harvesting complexes of photosystem II (PSII), is now generally accepted as a system that protects PSII by dynamically modulating energy transfer from the antennae to the reaction center, thus regulating the quantum efficiency of PSII (1-6). There is clear evidence from the effects of dark ATP-hydrolysis-induced acidification of thylakoids that this system is "energy dependent" and obviously independent of electron transport or associated redox changes induced by light (7). Under such conditions, a short-lifetime fluorescence component or putative quenching complex is formed (8).Zeaxanthin and antheraxanthin are formed from violaxanthin by violaxanthin de-epoxidase (VDE) activity in the socalled xanthophyll or violaxanthin cycle (9). De-epoxidationdependent energy dissipation is capable of responding to light environments over a wide dynamic range from sun-flecks to long-term growth conditions mainly due to the properties of the xanthophyll cycle (10, 11). The response range apparently gives higher plants the ability to adapt photosynthetically to diverse environments and, for this reason, may have be...

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

“…If, as is the case of the xanthophyll cycle de-epoxidase enzyme, activities of the carotene hydroxylases are pH-dependent, localization/orientation on the thylakoid membranes may be critical. At high light irradiance, acidification of the thylakoid lumen activates membrane association of the violaxanthin de-epoxidase, an enzyme having a pI of 4.57 [75], to convert violaxanthin to zeaxanthin, a product which dissipates the high light energy; in the dark violaxanthin accumulates. Perhaps diiron carotene β-ring hydroxylases function in the dark or on different plastid membranes (including envelope) for apocarotenoid biosynthesis, while P450 β-ring hydroxylases are light activated or function on the thylakoid membranes in association with the photosynthetic apparatus.…”

Abscisic Acid Biosynthesis and Response

“…Indeed, a mutant isolated on the basis of altered nonphotochemical quenching (npq2) is allelic to aba1 . The gene encoding the enzyme responsible for the reverse reaction, violaxanthin de-epoxidase, which is an important activity regulating the xanthophyll cycle, is encoded by the NPQ1 locus and was previously cloned from lettuce (Bugos and Yamamoto, 1996). The aba1 gene was first identified by virtue of the generation of a transposon-tagged, non-dormant wilty mutant of Nicotiana plumbaginifolia (Npaba2) that was shown to be orthologous to Arabidopsis aba1 .…”

A D1 protein mutáció hatása a kettes fotokémiai rendszer energiahasznosítására eltérő vízellátású növényekben

“…There is a general consensus that NPQ is controlled by factors that are encoded in the nucleus (Funk et al, 1995;Bugos & Yamamoto, 1996;Jansson, 1999;Li et al, 2000). Since the D1 protein encoding psbA gene, which carries the mutation in the atrazineresistant plants, is localized in the chloroplast genome (Morden & Golden, 1989), it is not entirely clear in what way the D1 protein can contribute to the formation of efficient nonphotochemical dissipative processes in the antenna.…”

“…A szakirodalom szerint az NPQ kialakulásában esszenciális faktorok sejtmagi gének által kódoltak (Funk et al, 1995;Li et al, 2000;Bugos & Yamamoto, 1996;Jansson, 1999), habár a D1 fehérjét kódoló psbA gén a kloroplasztisz genom része. Nem világos, hogy a D1 fehérje milyen hatással van az antennában végbemenő hődisszipáció kialakulására.…”

“…Elképzelhető, hogy a S. Az NPQ molekuláris mechanizmusa csak részben ismert, de kialakulásának feltételei jól behatárolhatóak: a tilakoid lumenének alacsony kémhatása, de-epoxidált xantofillok képződése, a PsbS és más LHCII fehérjék protonációja. Eddigi ismereteink szerint az NPQ kialakulását nukleárisan kódolt faktorok határozzák meg (Funk et al, 1995;Li et al, 2000;Bugos & Yamamoto, 1996;Jansson, 1999), habár a D1 fehérjét kódoló psbA gén -amely a rezisztens növényekben a mutációt hordozza -a kloroplasztisz genom része (Morden & Golden, 1989). Nem világos, hogy a D1 protein hogyan járulhat hozzá az antennában végbemenő, hatékony nem-fotokémiai hődisszipáció kialakulásához.…”