From methylglyoxal to pyruvate: a genome-wide study for the identification of glyoxalases and D-lactate dehydrogenases in Sorghum bicolor

Abstract

Background: The glyoxalase pathway is evolutionarily conserved and involved in the glutathione-dependent detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis. It acts via two metallo-enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), to convert MG into D-lactate, which is further metabolized to pyruvate by D-lactate dehydrogenases (D-LDH). Since D-lactate formation occurs solely by the action of glyoxalase enzymes, its metabolism may be considered as the ultimate step of MG detoxification. By maintaining steady state levels of MG and other reactive dicarbonyl compounds, the glyoxalase pathway serves as an important line of defence against glycation and oxidative stress in living organisms. Therefore, considering the general role of glyoxalases in stress adaptation and the ability of Sorghum bicolor to withstand prolonged drought, the sorghum glyoxalase pathway warrants an in-depth investigation with regard to the presence, regulation and distribution of glyoxalase and D-LDH genes.

Result: Through this study, we have identified 15 GLYI and 6 GLYII genes in sorghum. In addition, 4 D-LDH genes were also identified, forming the first ever report on genome-wide identification of any plant D-LDH family. Our in silico analysis indicates homology of putatively active SbGLYI, SbGLYII and SbDLDH proteins to several functionally characterised glyoxalases and D-LDHs from Arabidopsis and rice. Further, these three gene families exhibit development and tissue-specific variations in their expression patterns. Importantly, we could predict the distribution of putatively active SbGLYI, SbGLYII and SbDLDH proteins in at least four different sub-cellular compartments namely, cytoplasm, chloroplast, nucleus and mitochondria. Most of the members of the sorghum glyoxalase and D-LDH gene families are indeed found to be highly stress responsive.

Conclusion: This study emphasizes the role of glyoxalases as well as that of D-LDH in the complete detoxification of MG in sorghum. In particular, we propose that D-LDH which metabolizes the specific end product of glyoxalases pathway is essential for complete MG detoxification. By proposing a cellular model for detoxification of MG via glyoxalase pathway in sorghum, we suggest that different sub-cellular organelles are actively involved in MG metabolism in plants.

Keywords: D-lactate dehydrogenase; Genome-wide analysis; Glyoxalase; Sorghum; Stress.

Fig. 1

Schematic representation of the glyoxalase pathway for methylglyoxal detoxification in plants. Methylglyoxal (MG) is converted to S-D-lactoylglutathione (SLG) by glyoxalase I (GLYI) enzyme which is then converted to D-lactate by glyoxalase II (GLYII). Glutathione is used in the first reaction catalysed by GLYI but is recycled in the second reaction catalysed by GLYII. D-lactate is further metabolized to pyruvate through D-lactate dehydrogenase (D-LDH) enzyme which passes electrons to cytochrome C (CYTc)

Fig. 2

Phylogenetic analysis of glyoxalase proteins from sorghum and other plant species. Circular tree constructed for the (a) GLYI and (b) GLYII proteins from sorghum, rice, Arabidopsis, Medicago and Soybean using Neighbour-Joining method in MEGA 7.0 with 1000 bootstrap replicates. The putative sub-cellular localisation of the proteins has been indicated as rings bordering the tree in different colours. Cytoplasm (red), Chloroplast (green), Mitochondria (blue), Nucleus (purple), Extracellular/peroxisomes (yellow), Chloroplast or Mitochondria (turquoise). The localisation of those marked with asterisk have been experimentally proven

Fig. 3

Exon-intron organisation of glyoxalase gene family from sorghum. Exon-Intron structure of (a) SbGLYI and (b) SbGLYII genes were analysed using the Gene Structure Display Server tool. Length of exons and introns has been exhibited proportionally as indicated by the scale on the bottom. Order of GLY genes is represented as per their phylogenetic relationship. The branch lengths represent evolutionary time between the two nodes

Fig. 4

Schematic representation of domain architecture of glyoxalase proteins from sorghum. Domain architecture of (a) SbGLYI proteins showing the presence of glyoxalase domain (PF00903) and (b) SbGLYII proteins containing metallo-beta lactamase superfamily domain (PF00753) in all the predicted SbGLYII proteins. In addition, HAGH_C (PF16123) domain predicted to be important for the catalytic activity of SbGLYII proteins, was also found in some SbGLYII protein sequences while few SbGLYII proteins had other secondary domains. Domains were analysed using Pfam database. Exact position and number of domains are schematically represented along with the length of the protein

Fig. 5

Developmental and stress-mediated regulation of glyoxalase family genes from sorghum. Expression profile of (a) GLYI and (b) GLYII genes was obtained from the publicly available Genevestigator Affymetrix sorghum genome array database. Normalized transcript data was obtained for different tissues, viz. underground tissues - root and, aerial tissues - leaf, internode, shoot and pith (left panel) at different developmental stages (middle panel). Normalized and curated perturbation expression data (right panel) of the genes was retrieved from Expression Atlas. Fold change in expression pertaining to ABA treatment (20 μM), PEG treatment (20% PEG 8000) and nutrient nitrogen limitation has been shown as heatmap generated using MeV software package. Colour scale below or on the right of the heatmap shows the level of expression. GLY genes have been represented in order as per their phylogenetic relationship. Branch length represents evolutionary time between the two nodes. Histogram depicting relative expression levels of (c) SbGLYI and (d) SbGLYII genes under different abiotic stress treatments viz. heat, cold, salinity (given to 7 d old seedlings for 6 h) and drought (water withheld for 48 h). Expression levels have been calculated with respect to the untreated control (having value of 1)

Fig. 6

Elucidation of exon-intron structure, protein domain architecture and phylogenetic relationship between sorghum D-LDH proteins. a Exon-Intron structure of SbDLDH genes. Length of exons and introns have been represented proportionally as indicated by the scale at the bottom. b Schematic representation of domain architecture of SbDLDH proteins indicating the presence of FAD_binding_4 and FAD_oxidase_C domains in SbDLDH proteins c Full length amino acid sequence of SbDLDH proteins were compared with the known D-LDH proteins from rice and Arabidopsis and phylogenetic tree was constructed using the Neighbour-Joining method in MEGA 7.0 with 1000 bootstrap replicates. Putative sub-cellular localisation of proteins have been indicated towards the right of the tree in different colours; cytoplasm (red) and mitochondria (blue)

Fig. 7

Developmental and stress-mediated regulation of D-LDH genes from sorghum. Genome-wide microarray data of D-LDH genes was obtained from the publicly available Genevestigator Affymetrix sorghum genome array database. Normalized transcript data was obtained for (a) different tissues, including underground tissues-root and, aerial tissues- shoot, leaf, internode and pith and at (b) different developmental stages. c Normalized and curated perturbation expression data of the sorghum D-LDH genes was retrieved from Expression Atlas. Fold change in expression pertaining to ABA treatment (20 μM), PEG treatment (20% PEG 8000) and nutrient nitrogen limitation has been shown as heatmap generated using MeV software package. Colour scale below and on the right of the heatmap shows the levels of expression. Genes have been represented in order as per their phylogenetic relationship. Branch length represents evolutionary time between the two nodes. d Histogram depicting relative expression levels of SbDLDH genes under different abiotic stress treatments viz. heat, cold, salinity, (given to 7 d old seedlings for 6 h) and drought (water withheld for 48 h). Expression levels have been calculated with respect to the untreated control (having a value of 1)

Fig. 8

Three dimensional structure of putative D-LDH proteins from sorghum generated through homology modelling. Three dimensional structures of putative D-LDH proteins were modelled using Rhodopseudomonas palostris (RhoPaDH) putative dehydrogenase (RhoPADH) (a) as template. Structures of (b) SbDLDH-1, (c) SbDLDH-2, (d) SbDLDH-3 and (e) SbDLDH-4 showing conserved FAD binding sites (marked in pink). SbDLDH-1 and SbDLDH-2, closest in structural similarity to RhoPaDH, are shown as overlay with RhoPaDH (f&g). Three-dimensional structure of D-LDH from E. coli (h) has also been shown as overlay with SbDLDH-1 (i) and SbDLDH-2 (j). Red indicates FAD binding site in E. coli, blue indicates catalytic site of E. coli D-LDH

Fig. 9

Proposed model of methylglyoxal detoxification via glyoxalase pathway proteins in various subcellular organelles of sorghum. Cellular defence against MG probably involves four different sub-cellular compartments viz. cytosol, chloroplast, mitochondria and nucleus. Cytosolic MG produced as an off shoot of glycolysis is converted to SLG by SbGLYI-10/11 which is further converted to D-lactate by SbGLYII-3. The conversion of D-lactate to pyruvate is catalysed either by SbDLDH-3, 4.1 or 4.2. Both in the mitochondria as well as chloroplast, MG detoxification is predicted to be catalysed by the same SbGLYI and SbGLYII proteins. D-lactate produced in the chloroplast can be converted to pyruvate either by cytosolic SbDLDH protein or transported in the mitochondria. In mitochondria, D-lactate is converted to pyruvate by SbDLDH-1/2 protein. Pyruvate is then fed into the Kreb’s cycle. In the nucleus, SbGLYI-8/SbGLYI-8.1, may catalyse the conversion of MG to SLG. Nuclear export of SLG is proposed as no nuclear GLYII could be predicted in the sorghum genome. TPI-Triose phosphate isomerase, GSH-Glutathione, G3P-Glyceraldehyde-3-Phosphate, F-1,-6-BP- Fructose-1,6-bisphosphate, Ru-1,5-BP- Ribulose-1,5, bisphosphate, PGA- Phosphoglyceraldehyde