A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize

Abstract

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.

Figure 1

Phylogenetic tree showing relationship of all known maize GST protein sequences. The horizontal scale shows the number of differences per 100 residues derived from the Clustal V alignment. Details of construction are in “Materials and Methods” and NCBI accession numbers are shown in Table I.

Figure 2

Phylogenetic tree showing relationship of all known soybean GST protein sequences. The horizontal scale shows the number of differences per 100 residues derived from the Clustal V alignment. Details of construction are in “Materials and Methods” and NCBI accession numbers are shown in Table II.

Figure 3

A bar graph showing the distribution of type I, type II, and type III GSTs within maize and soybean.

Figure 4

Expression of maize GST genes by DNA microarray analysis. The names of the new GSTs presented in this study have been abbreviated so that only the numerical designation is shown (e.g. ZmGST 13 is 13). A, Relative intensity values for each gene spot after hybridization to Cy3 labeled cDNA probe prepared from mRNA isolated from untreated etiolated maize seedlings. B, Fold induction of GST genes due to treatment of etiolated maize seedlings with 5 μL L⁻¹ dichlormid. Fold induction equals the intensity value for each gene target with a Cy5 labeled cDNA probe derived from treated tissue divided by the intensity value for each gene target with a Cy3-labeled cDNA probe derived from untreated tissue. C, Fold induction of GST genes due to treatment of etiolated maize seedlings with 10% (v/v) ethanol.

Figure 5

The consensus sequence report of all known maize and soybean GSTs. The amino acid sequences in each of the three classes of GSTs were aligned using the Clustal V method in the Megalign module of DNASTAR. Only the consensus sequence is shown. The histograms show the relative abundance of each amino acid in the group of sequences according to the consensus strength score that is a whole number from 0 to 5 (0, no bar; 1, dark blue; 2, light blue; 3, green; 4, orange; and 5, red). The red arrows indicate the three amino acids that are totally conserved in all of the GSTs reported here. A, The consensus report from all known maize and soybean type I GSTs. Note that a single sequence ZmGST 16 at a length of 299 amino acids is substantially longer than all of the other GSTs and accounts for the long stretch of unique sequence at the C terminus of the consensus. B, The consensus report from all known maize and soybean type II GSTs. Note that there are only three members of this class and they are quite closely related. C, The consensus report from all known maize and soybean type III GSTs.

Figure 6

Three-dimensional structure of plant GSTs with the strongly conserved type III features mapped. The active site Ser is shown in green as a space-filled model. The conserved patches in the Type III consensus sequence are shown as ribbons and colored as red, S20-L38; blue, K49-H68; orange, E76-E86; and yellow, L101-W114. A, The lactoylglutathione complex of a GSTI dimer taken from Neuefeind et al. (1997a). The substrate analog is shown as a space-filled model using Corey, Pauling, and Koltun colors. The regions of GSTI that are homologous to the type III conserved patches are S11-E29 (red), K41-N58 (blue), E66-R76 (orange), and R84-W98 (yellow). B, A homology model of ZmGST 24 prepared as described in the text. The monomer is shown in the same orientation as the GSTI dimer. The conserved patches in the ZmGST 24 sequence are S11-E29 (red), K38-H57 (blue), E64-E74 (orange), and L85-W98 (yellow).