Anthony H.C. Huang

Accumulation of proline and glycinebetaine in Spartina alterniflora Loisel. in response to NaCl and nitrogen in the marsh

SummaryThe possible interaction of high soil salinity and low soil nitrogen content in affecting the growth of Spartina alterniflora Loisel in the high and low marshes of the Eastern U.S. was explored. Throughout the whole growing season, the short plants growing in the high marsh, where there was a higher soil salinity and lower available soil nitrogen, contained more proline and glycinebetaine and showed a lower leaf water potential than the tall plants growing in the low marsh. In both short and tall plants, the growing season, with the highest content occurring in spring and fall. In contrast, the glycinebetaine content in both short and tall plants remained fairly constant throughout the growing season, and was consistently at least 10 fold higher than the proline content. It is estimated that 19–30% of the total leaf nitrogen was in the form of proline and glycinebetaine in the short plants, and 14–27% in the tall plants. Ammonium nitrate fertilization in the field resulted in increased growth, higher proline and glycinebetaine contents, and lower water potentials in the short plants, but had little effect on these parameters in the tall plants. We suggest that in the low marsh, the plants can obtain sufficient nitrogen for osmoregulation and other metabolism. In the high marsh with higher soil salinity and lower nitrogen content, the plants have to allocate a even greater proportion of the already limited nitrogen supply for osmoregulation. Thus, nitrogen available for osmoregulation and other nitrogen-requiring metabolism is insufficient, resulting in reduced growth.

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napus L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl2, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.

Lipases in the storage tissues of peanut and other oil seeds during germination.

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.

Transport of glycine, serine, and proline into spinach leaf mitochondria

The transport of radioactive glycine, serine, and proline into the matrix of spinach leaf mitochondria was studied using the silicone oil centrifugation technique. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mM, an apparent diffusion process dominated. The uptake was not saturable at increasing amino acid concentrations, and there was no accumulation of amino acid in the matrix (i.e., concentration was similar to that in the medium). At concentrations lower than 0.5 mM, in addition to the diffusion process, an active uptake system that accumulated amino acid in the matrix became apparent. This system was partially inhibited by rotenone, antimycin A, and carbonylcyanide-m-chlorophenyl hydrazone. Also, uptake of glycine and serine was mutually inhibitory. These two amino acids exhibited comparatively less inhibitory effect on proline uptake, and proline also did not inhibit glycine or serine uptake. The results suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity. The diffusion process dominates at amino acid concentrations of 0.5 mM or higher, whereas the active uptake system becomes more prominent as the amino acid concentration decreases.

Characteristics and biosynthesis of seed lipases in maize and other plant species

Oilseed lipases from diverse plant species exhibit differences in their substrate specificity, pH for optimal activity, reactivity toward sulfhydryl reagents, hydrophobicity and subcellular location. Seed lipase from a certain plant species is relatively specific for the native triacylglycerols or triacylglycerols containing the major fatty acids of the storage triacylglycerols of the same species. This substrate specificity can be exploited in lipid biotechnology. In most seeds, with the known exception of castor bean, lipase activities are absent in ungerminated seeds and increase in postgermination. The biosynthesis of seed lipase has been studied only in maize. The maize enzyme is synthesized on free polyribosomes in postgermination. The newly synthesized enzyme is then transferred, without apparent coor posttranslational modification, to the membrane of the lipid bodies.

Conversion of serine to glycerate in intact spinach leaf peroxisomes: Role of malate dehydrogenase

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 M sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 mM serine, but was two to three times higher in 50 mM than 5 mM malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 mM glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-alpha-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limiting step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD/NADH ratio in the cytosol.

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings

Abstract During the germination of jojoba ( Simmondsia chinensis ) seeds, fatty alcohols are formed from the hydrolysis of stored wax esters. The cotyledon extract has the ability to convert fatty alcohols to fatty aldehydes in the presence of molecular oxygen and subsequently to fatty acids when NAD+ is added. The two enzymes which catalyze these activities have been partially characterized. The fatty alcohol oxidase utilizes molecular oxygen as the electron acceptor and dodecyl alcohol as the preferred substrate and has an optimal pH for activity at 9.0. The fatty aldehyde dehydrogenase also has an optimal pH for activity at 9.0, an apparent K m value of 4 × 10 −6 m for decyl aldehyde, an apparent K m value of 2.5 × 10 −4 m for NAD+, and a substrate preference for dodecyl aldehyde. NAD+ is a much better electron acceptor than NADP+, FAD, or flavin mononucleotide for the aldehyde dehydrogenase. Both enzyme activities are inhibited by p -chloromercuribenzoate and its effect is completely reversed by dithiothreitol. The whole fatty alcohol oxidation system is capable of oxidizing monounsaturated fatty alcohols which are the physiological substrates in jojoba cotyledons. The two enzyme activities are absent in the dry seed and increase drastically during germination. Both enzymes are localized primarily on the membrane of the wax bodies, although they may be present in other cellular membranes.

Conversion of glycerate to serine in intact spinach leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes converted glycerate to serine in the presence of NAD and alanine. The reaction proceeded optimally at pH9. Addition of oxaloacetate or alpha-ketoglutarate plus aspartate enhanced the conversion about three-fold. Alteration of the concentration of one of the reaction components, consisting of 2 mM glycerate, 0.2 mM NAD, 0.5 mM oxaloacetate, and 2 mM alanine, revealed half-saturation constants of 0.45 mM for glycerate, 0.06 mM for NAD, 0.02 mM for oxaloacetate, and 0.33 mM for alanine. The conversion proceeded with the formation of hydroxypyruvate followed by serine; hydroxypyruvate did not accumulate to a high amount in the presence or absence of alanine. The amino group donor could be alanine (half-saturation constant, 0.33 mM), glycine (0.45 mM), or asparagine (0.67 mM); the three amino acids produced roughly similar Vmax values. The results indicate that, in the conversion of glycerate to serine, the transamination is catalyzed by a hydroxypyruvate aminotransferase with characteristics unknown among all other studied leaf peroxisomal aminotransferases. The peroxisomal membrane is sparsely permeable to NAD/NADH, and the participation of the peroxisomal malate dehydrogenase in an electron shuttle system across the membrane in the regeneration of NAD/NADH is suggested.

Glyoxysomal acyl-CoA synthetase and oxidase from germinating elm, rape and maize seed

Abstract The activities of acyl-CoA synthetase (EC 6.2.1.3; 3) and acyl-CoA oxidase (EC 1.3.99.3; 4) in the glyoxysomes from germinating elm, maize and rape see

Developmental studies of NAD-malate dehydrogenase isozymes in the cotyledon of cucumber seedlings grown in darkness and in light

SummaryThe total activity of NAD-malate dehydrogenase (E.C. 1.1.1.37; MDH) in the cotyledons of cucumber (Cucumis sativus L.) seeds and seedlings increased 10fold during the first 3 days of germination in darkness and then declined gradually to one third of the peak activity after 10 days. Exposure of the seedlings to light at day 3 sharply reduced the activity to one third of the peak activity within 1 day but the residual activity remained fairly constant from day 4 to day 10. In extracts of cotyledons of 2-day-old, dark-grown seedlings and of cotyledons of 6-day-old, light-grown seedlings MDH activity was resolved into 5 isozymes by starch gel electrophoresis. Three of these isozymes were identified as cytosol isozymes, one each as a mitochondrial isozyme and a microbody (glyoxysome or peroxisome) isozyme. During germination in darkness, the activities of the five isozymes increased at different rates. However, they all peaked together at day 3 and then declined gradually at a similar rate. Light applied at day 3 selectively eliminated two cytosol isozymes but did not affect the subsequent developmental pattern of the third cytosol isozyme, the mitochondrial isozyme and the microbody isozyme. Such a selective elimination of two cytosol isozymes, together with their absence in roots and green leaves, indicates that these two isozymes participate in the mobilization of food reserves during germination. The glyoxysomal and peroxisomal isozymes were not resolved in starch gel electrophoresis. Their similarities are discussed in connection with the relationship of the two enzymes and of the two types of microbodies during the greening process.