Betaine Aldehyde Oxidation by Spinach Chloroplasts

Abstract

Chenopods synthesize betaine by a two-step oxidation of choline: choline -. betaine aldehyde -. betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H202-mediated reaction cannot be ruled out.Betaine (glycinebetaine) accumulates in response to salinization or to water deficit in chenopods, grasses, and in other angiosperm families (8) as well as in a number of prokaryotes (11,14). For higher plants, Wyn Jones et al. (30) have proposed that betaine is localized mainly in the cytoplasm, where it acts as a nontoxic osmoticum, allowing osmotic adjustment to occur without perturbing metabolic functions. Much evidence (7) now supports this proposal, which accords betaine synthesis a major role in adaptation to osmotic stress.In-vivo radiotracer studies (8) show that betaine is synthesized in leaves from a two-step oxidation of choline:Little is known about the nature of these reactions in plants or about the enzymes involved. However, the enzymology of choline oxidation is quite well known for mammalian liver, in which both steps are mitochondrial (10, 29), and for certain microorganisms (15-17). In these nonplant systems the choline -+ betaine aldehyde step is catalyzed by a flavoprotein dehydrogenase or oxidase, the betaine aldehyde --betaine step by a specific 'Funded We recently showed that both steps in choline oxidation are chloroplastic in spinach, that they are light-promoted, and that the effect of light is sensitive to DCMU (9). On the other hand, Pan et al. (19) reported that the cytosolic fraction from spinach leaves contained NAD-dehydrogenase activity specific for betaine aldehyde, and that the chloroplast fraction lacked this activity. Therefore, in this work we examined (a) the mechanisms by which spinach chloroplasts oxidize betaine aldehyde in darkness and light, and (b) the subcell distributio...

Choline-O-Sulfate Biosynthesis in Plants (Identification and Partial Characterization of a Salinity-Inducible Choline Sulfotransferase from Species of Limonium (Plumbaginaceae)

“…Salinization with ASW induced CST in roots, leaves, and cell cultures by up to 6-fold. This induction is consistent with the 2-to %fold increase in CS content in salinized plants (Hanson et al, 1991) and is of similar magniiude to that found for the enzymes of Gly betaine synthesis in salinized spinach (Weigel et al, 1986;Brouquisse et al, 19f19;Summers and Weretilnyk, 1993). Because ASW contains 110th C1-and SO4'-(in a 19:l ratio), our salinization data d.o not show whether CST induction is a specific response to !$O4*-.…”

Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays)

“…Reliable controls include observations in the wild type and YFP alone transgenic plants that argue against other possibilities of the predicted subcellular localizations. To our knowledge, characterized plant ALDHs have been demonstrated to be targeted to diverse compartments including mitochondria (Busch and Fromm 1999;Nakazono et al 2000;Deuschle et al 2001;Liu et al 2001;Skibbe et al 2002), plastid/chloroplast (Kirch et al 2001;Weigel et al 1986;Kotchoni et al 2006), Cytosol (Rumpho et al 1983Nair et al 2004;Kotchoni et al 2006), and peroxisome (Nakamura et al 1997). Although the biochemical function of the aldehyde dehydrogenase characterized here is not yet clear, physiological properties suggest a detoxification function in plastids.…”

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

“…BADH was precipitated from the supernatant with (NH,),SO, (35-70% saturation), resuspended in 50 mM Tris-HCI, pH 7.5, desalted on Pharmacia PD-10 columns equilibrated in the same buffer, flash frozen in 0.1-mL aliquots in liquid nitrogen, and stored at -80°C. The BADH activity of the preparations, measured spectrophotometrically in 50 mM Hepes-KOH, pH 8.0 (Weigel et al, 1986), was 27 pkat pL-'; choline-oxidizing activity measured in the assay given below was negligible.…”