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NADPH supply and mannitol biosynthesis. Characterization, cloning, and regulation of the non-reversible glyceraldehyde-3-phosphate dehydrogenase in celery leaves - PubMed

NADPH supply and mannitol biosynthesis. Characterization, cloning, and regulation of the non-reversible glyceraldehyde-3-phosphate dehydrogenase in celery leaves

Z Gao et al. Plant Physiol. 2000 Sep.

Abstract

Mannitol, a sugar alcohol, is a major primary photosynthetic product in celery (Apium graveolens L. cv Giant Pascal). We report here on purification, characterization, and cDNA cloning of cytosolic non-reversible glyceraldehyde-3-P dehydrogenase (nr-G3PDH, EC 1.2.1. 9), the apparent key contributor of the NADPH required for mannitol biosynthesis in celery leaves. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified nr-G3PDH showed a molecular mass of 53 kD. A 1,734-bp full-length cDNA clone (accession no. AF196292) encoding nr-G3PDH was identified using polymerase chain reaction and rapid amplification of cDNA ends techniques. The cDNA clone has an open reading frame of 1,491 bp encoding 496 amino acid residues with a calculated molecular weight of 53,172. K(m) values for the celery nr-G3PDH were low (6.8 microM for NADP(+) and 29 microM for D-glyceraldehyde-3-P). NADPH, 3-phosphoglycerate, and ATP were competitive inhibitors, and cytosolic levels of these three metabolites (as determined by nonaqueous fractionation) were all above the concentrations necessary to inhibit activity in vitro, suggesting that nr-G3PDH may be regulated through feedback inhibition by one or more metabolites. We also determined a tight association between activities of nr-G3PDH and mannose-6-P reductase and mRNA expression levels in response to both leaf development and salt treatment. Collectively, our data clearly show metabolic, developmental, and environmental regulation of nr-G3PDH, and also suggest that the supply of NADPH necessary for mannitol biosynthesis is under tight metabolic control.

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Figures

Figure 1
Figure 1

nr-G3PDH activity in developing celery leaves and the effect of salt on the activities. Celery plants were grown with 0 (control), 50, 150

,

and 300 m

m

NaCl in the irrigation solutions for 15 d prior to sampling.

Figure 2
Figure 2

An SDS-PAGE analysis showing the various stages of purification of nr-G3PDH from celery leaves. Lane M, Molecular mass standards (Bio-Rad Laboratories, broad range); lane 1, after acetone precipitation (20 μg); lane 2, pooled active fractions from a DEAE-Sepharose column (20 μg); lane 3, pooled active fractions from a Sephadex-200 column (20 μg); lane 4, pooled active fractions from an Affi-Blue Sepharose column (20 μg). Proteins were stained with Coomassie Blue. Molecular mass of nr-G3PDH is indicated on the right side of the gel as 53 kD.

Figure 3
Figure 3

Inhibition of nr-G3PDH activity by NADPH and 3-PGA. Assays were performed by varying NADP+ concentrations at a fixed D-G3P (100 μ

m

) with addition of 0, 10, 20

,

or 40 μ

m

NADPH (A), by varying D-G3P concentrations at a fixed NADP+ (100 μ

m

) with addition of 0, 1.25, 2.5

,

or 5 m

m

3-PGA (B), and by varying NADP+ concentrations at a fixed D-G3P (100 μ

m

) with addition of 0, 1.25, or 2.5 m

m

3-PGA (C).

Figure 4
Figure 4

Competitive inhibition by ATP against NADP+ (A) and D-G3P (B). Assays were performed by varying NADP+ or D-G3P with a fixed concentration (100 μ

m

) of the other substrate. ATP concentrations (m

m

) in the assays are indicated. The apparent competitive inhibition shifted toward a mixed-type inhibition at ATP concentrations >2.5 m

m

, but these are probably not physiologically important and thus are not shown.

Figure 5
Figure 5

Distribution of marker enzymes from fractions derived from a nonaqueous density gradient of freeze-dried, sonicated celery leaves. Typically, distribution of α-mannosidase (EC 3.2.1.24) is in the vacuole, NADP-dependent reversible glyceraldehyde-3-P dehydrogenase (GAP-DH, EC 1.2.1.13) is in the chloroplast, and PEP carboxylase (PEPcase, EC 4.1.1.31) is in the cytosol. Recoveries of the marker enzymes were in the range of 93% to 106% based on total activities in the unfractionated materials.

Figure 6
Figure 6

Sequence alignment of celery nr-G3PDH with other sequences. The deduced amino acid sequence of celery nr-G3PDH (a) aligned with the nr-G3PDH gene from tobacco (b; accession no. U87848), the nr-G3PDH gene from pea (c; Habenicht et al., 1994), a putative nr-G3PDH peptide from Arabidopsis (d; accession no. AC005967), and the nr-G3PDH gene from maize (e; Habenicht et al., 1994). The amino acids used for the degenerate primers to obtain the 900-bp fragment are underlined. The three most conserved regions present in all non-phosphorylating ALDHs are marked with asterisks. Cys-298, indicated by the arrow, is postulated to be involved in the formation of the thioacylenzyme intermediate for all ALDH enzymes (Lindahl, 1992; Habenicht et al., 1994).

Figure 7
Figure 7

A, Northern analysis of nr-G3PDH mRNA from total RNA (10 μg per lane) extracted from young leaves (Y), immature leaves (IM), mature leaves (M), and senescent yellowish leaves (S) of celery plants. B, Northern analysis of nr-G3PDH mRNA from total RNA (10 μg per lane) extracted from mature leaves of celery plants treated with 0, 50, 100, 150, and 300 m

m

NaCl. The blots were probed with a 900-bp PCR fragment of celery nr-P3PDH as described in “Materials and Methods.” The loading of each lane was verified by reprobing the blot with an 18S rRNA probe from Arabidopsis.

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