David J. Oliver

Background: Eukaryotic aldehyde dehydrogenases (ALDHs, EC 1.2.1), which oxidize aldehydes into carboxylic acids, have been classified into more than 20 families. In mammals, Family 2 ALDHs detoxify acetaldehyde. It has been hypothesized that plant Family 2 ALDHs oxidize acetaldehyde generated via ethanolic fermentation, producing acetate for acetyl-CoA biosynthesis via acetyl-CoA synthetase (ACS), similar to the yeast pathway termed the "pyruvate dehydrogenase (PDH) bypass". Evidence for this pathway in plants has been obtained from pollen. Results: To test for the presence of the PDH bypass in the sporophytic tissue of plants, Arabidopsis plants homozygous for mutant alleles of all three Family 2 ALDH genes were fed with ¹⁴C-ethanol along with wild type controls. Comparisons of the incorporation rates of ¹⁴C-ethanol into fatty acids in mutants and wild type controls provided direct evidence for the presence of the PDH bypass in sporophytic tissue. Among the three Family 2 ALDHs, one of the two mitochondrial ALDHs (ALDH2B4) appears to be the primary contributor to this pathway. Surprisingly, single, double and triple ALDH mutants of Arabidopsis did not exhibit detectable phenotypes, even though a Family 2 ALDH gene is required for normal anther development in maize. Conclusion: The PDH bypass is active in sporophytic tissue of plants. Blocking this pathway via triple ALDH mutants does not uncover obvious visible phenotypes.

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the γ-glutamyl cycle, where GSH is degraded by the sequential reaction of γ-glutamyl transpeptidase (GGT), γ-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that γ-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of γ-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous ¹⁴C-acetate and ¹⁴C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.

Summary: γ‐Glutamyl transpeptidase (GGT) is the enzyme responsible for breaking the γ‐glutamyl bond between Glu and Cys in glutathione (GSH). We are using this gene family to study GSH degradation in plants. There are four putative GGT genes in Arabidopsis, and one of them, GGT3 (At4g29210), is analyzed in this study. GGT3 is localized to the vacuole based on organelle‐targeting programs, subcellular distribution of GFP fusion proteins during transient expression in onion (Allium cepa) epidermal tissues, and its ability to metabolize vacuolar substrates in Arabidopsis plants. While Northern blots and promoter:GUS expression patterns have suggested that GGT3 is transcribed at relatively high levels in all parts of the plant, a comparison of enzyme activities in different organs of wild‐type and a ggt3 knockout mutant showed that GGT3 was a major contributor to total GGT activity in roots, but a relatively minor contributor in other tissues. Wild‐type Arabidopsis plants treated with monobromobimane (mBB) form a fluorescent GSH–mBB conjugate that is moved into the vacuole and then metabolized to Cys‐Gly–mBB and Cys–mBB in that order. The first step is catalyzed by GGT3, and GSH–mBB metabolism is completely blocked in the roots of ggt3 knockout plants. In ggt3 leaves, some GSH–mBB metabolism still proceeds using the apoplastic GGT1. This identifies GGT3 as catalyzing the obligate initial step in GSH conjugate metabolism, and suggests that it has an important role in protecting plants from some xenobiotic chemicals.

Summary: γ‐Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the γ‐peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid‐rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild‐type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.

Activation sequence-1 (as-1) cognate promoter elements are widespread in the promoters of plant defense-related genes as well as in plant pathogen promoters, and may play important roles in the activation of defense-related genes. The as-1-type elements are highly responsive to multiple stress stimuli such as jasmonic acid (JA), salicylic acid (SA), H(2)O(2), xenobiotics and heavy metals, and therefore provide a unique opportunity for identifying additional signaling components and cross-talk points in the various signaling networks. A single as-1-type cis-element-driven GUS reporter Arabidopsis line responsive to JA, SA, H(2)O(2), xenobiotics and heavy metals was constructed for mutagenesis. A large-scale T-DNA mutagenesis has been conducted in the reporter background, and an efficient high-throughput mutant screen was established for isolating mutants with altered responses to the stress chemicals. A number of mutants with altered stress responses were obtained, some of which appear to identify new components in the as-1-based signal transduction pathways. We characterized a mutant (Delta8L4) with a T-DNA insertion in the coding sequence of the gene At4g24275. The as-1-regulated gene expression and GUS reporter gene expression were altered in the Delta8L4 mutant, but there was no change in the expression of genes lacking as-1 elements in their promoters. The phenotype observed with the Delta8L4 mutant was further verified using RNAi plants for At4g24275 (8L4-RNAi), suggesting the feasibility of use of this high-throughput mutant screening in isolating stress-signaling mutants.

In contrast to peas (Pisum sativum), where mitochondrial lipoamide dehydrogenase is encoded by a single gene and shared between the alpha-ketoacid dehydrogenase complexes and the Gly decarboxylase complex, Arabidopsis has two genes encoding for two mitochondrial lipoamide dehydrogenases. Northern-blot analysis revealed different levels of RNA expression for the two genes in different organs; mtLPD1 had higher RNA levels in green leaves compared with the much lower level in roots. The mRNA for mtLPD2 shows the inverse pattern. The other organs examined showed nearly equal RNA expressions for both genes. Analysis of etiolated seedlings transferred to light showed a strong induction of RNA expression for mtLPD1 but only a moderate induction of mtLPD2. Based on the organ and light-dependent expression patterns, we hypothesize that mtLPD1 encodes the protein most often associated with the Gly decarboxylase complex, and mtLPD2 encodes the protein incorporated into alpha-ketoacid dehydrogenase complexes. Due to the high level of sequence conservation between the two mtLPDs, we assume that the proteins, once in the mitochondrial matrix, are interchangeable among the different multienzyme complexes. If present at high levels, one mtLPD might substitute for the other. Supporting this hypothesis are results obtained with a T-DNA knockout mutant, mtlpd2, which shows no apparent phenotypic change under laboratory growth conditions. This indicates that mtLPD1 can substitute for mtLPD2 and associate with all these multienzyme complexes.