In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase

Abstract

NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes.

Figure 1

Functions of the endogenous NADPH in plant peroxisomes. NADPH is required for several enzymatic systems including the glutathione reductase (GR) to keep the level of reduced glutathione (GSH), the L-arginine-dependent nitric oxide synthase (NOS) which generates nitric oxide (NO), and the 2,4-dienoyl-CoA reductase (DECR) which is necessary for the degradation of fatty acids unsaturated on odd-numbered carbons. 6PGDH: 6-phosphogluconate dehydrogenase. 6PG: 6-phosphogluconate. Rib 5-P: ribulose 5-phosphate.

Figure 2

Evolutionary relationships of plant 6PGDHs. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 2,42428701 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). The analysis involved 51 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 416 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [30].

Figure 3

Peroxisomal 6PGDH gene (At3g02360) expression in Arabidopsis thaliana grown under different conditions. (a) Expression of p6PGDH in leaves of 15-day-old plants, stems and flowers of 29-day-old plants grown in either growth chamber or greenhouse conditions. (b) Effects of a 6 h long treatment with 90 mM sucrose to 4-day-old, dark-grown Arabidopsis seedlings. (c) Effects of 5 μM norflurazon to 5-day-old, continuous light Arabidopsis seedlings. (d) Auxin effect on 10-day-old seedlings treated for 8 h either 0.1 μM 2,4-D or 0.1 μM 2,4-D plus 1 μM brassinazole. Data were obtained from the Gene Expression Omnibus (GEO) database and analyzed using Affymetrix Microarray Suite 5.0 (MAS5). The original sample accessions (GSMxxx) are listed in the gray boxes along the bottom of the chart.