Targeted quantitative profiling of metabolites and gene transcripts associated with 4-aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses.

4-Aminobutyrate accumulates in plants under abiotic stress. Here, targeted quantitative profiling of metabolites and transcripts was conducted to monitor glutamate- and polyamine-derived 4-aminobutyrate production and its subsequent catabolism to succinate or 4-hydroxybutyrate in apple (Malus x domestica Borkh.) fruit stored at 0 °C with 2.5 kPa O2 and 0.03 or 5 kPa CO2 for 16 weeks. Low-temperature-induced protein hydrolysis appeared to be responsible for the enhanced availability of amino acids during early storage, and the resulting higher glutamate level stimulated 4-aminobutyrate levels more than polyamines. Elevated CO2 increased the levels of polyamines, as well as succinate and 4-hydroxybutyrate, during early storage, and 4-aminobutyrate and 4-hydroxybutyrate over the longer term. Expression of all of the genes likely involved in 4-aminobutyrate metabolism from glutamate/polyamines to succinate/4-hydroxybutyrate was induced in a co-ordinated manner. CO2-regulated expression of apple GLUTAMATE DECARBOXYLASE 2, AMINE OXIDASE 1, ALDEHYDE DEHYDROGENASE 10A8 and POLYAMINE OXIDASE 2 was evident with longer term storage. Evidence suggested that respiratory activities were restricted by the elevated CO2/O2 environment, and that decreasing NAD+ availability and increasing NADPH and NADPH/NADP+, respectively, played key roles in the regulation of succinate and 4-hydroxybutyate accumulation. Together, these findings suggest that both transcriptional and biochemical mechanisms are associated with 4-aminobutyrate and 4-hydroxybutyrate metabolism in apple fruit stored under multiple abiotic stresses.

Corrigendum: Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

This corrects the article DOI: 10.1038/srep35115.

Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2) are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh.), rice (Oryza sativa L.) and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L.) suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and ß-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

NAD(+)-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and ß-alanine production during terminal oxidation of polyamines in apple fruit.

The last step of polyamine catabolism involves the oxidation of 3-aminopropanal or 4-aminobutanal via aminoaldehyde dehydrogenase. In this study, two apple (Malus x domestica) AMADH genes were selected (MdAMADH1 and MdAMADH2) as candidates for encoding 4-aminobutanal dehydrogenase activity. Maximal activity and catalytic efficiency were obtained with NAD(+) and 3-aminopropanal, followed by 4-aminobutanal, at pH 9.8. NAD(+) reduction was accompanied by the production of GABA and ß-alanine, respectively, when 4-aminobutanal and 3-aminopropanal were utilized as substrates. MdAMADH2 was peroxisomal and MdAMADH1 cytosolic. These findings shed light on the potential role of apple AMADHs in 4-aminobutyrate and ß-alanine production.

Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1 prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes monoamines.

4-Aminobutyrate (GABA) accumulates in apple fruit during controlled atmosphere storage. A potential source of GABA is the polyamine putrescine, which can be oxidized via copper-containing amine oxidase (CuAO), resulting in the production 4-aminobutanal/Δ(1)-pyrroline, with the consumption of O2 and release of H2O2 and ammonia. Five putative CuAO genes (MdAO genes) were cloned from apple (Malus domestica Borkh. cv. Empire) fruit, and the deduced amino acid sequences found to contain the active sites typically conserved in CuAOs. Genes encoding two of these enzymes, MdAO1 and MdAO2, were highly expressed in apple fruit and selected for further analysis. Amino acid sequence analysis predicted the presence of a C-terminal peroxisomal targeting signal 1 tripeptide in MdAO1 and an N-terminal signal peptide and N-glycosylation site in MdAO2. Transient expression of green fluorescent fusion proteins in Arabidopsis protoplasts or onion epidermal cells revealed a peroxisomal localization for MdAO1 and an extracellular localization for MdAO2. The enzymatic activities of purified recombinant MdAO1 and MdAO2 were measured continuously as H2O2 production using a coupled reaction. MdAO1 did not use monoamines or polyamines and displayed high catalytic efficiency for 1,3-diaminopropane, putrescine and cadaverine, whereas MdAO2 exclusively utilized aliphatic and aromatic monoamines, including 2-phenylethylamine and tyramine. Together, these results indicate that MdAO1 may contribute to GABA production via putrescine oxidation in the peroxisome of apple fruit under controlled atmosphere conditions. MdAO2 seems to be involved in deamination of 2-phenylethylamine, which is a step in the biosynthesis of 2-phenylethanol, a contributor to fruit flavor and flower fragrance.

Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit.

BACKGROUND: The ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O2 deficiency and elevated CO2. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca(2+)/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity. RESULTS: GAD activity in cell-free extracts of apple fruit was stimulated by Ca(2+)/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca(2+)/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca(2+)/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain. CONCLUSIONS: Plant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30-50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.

A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant.

BACKGROUND: Taxol is an anti-cancer drug harvested from Taxus trees, proposed ecologically to act as a fungicide. Taxus is host to fungal endophytes, defined as organisms that inhabit plants without causing disease. The Taxus endophytes have been shown to synthesize Taxol in vitro, providing Taxus with a second potential biosynthetic route for this protective metabolite. Taxol levels in plants vary 125-fold between individual trees, but the underlying reason has remained unknown. RESULTS: Comparing Taxus trees or branches within a tree, correlations were observed between Taxol content, and quantity of its resident Taxol-producing endophyte, Paraconiothyrium SSM001. Depletion of fungal endophyte in planta by fungicide reduced plant Taxol accumulation. Fungicide treatment of intact plants caused concomitant decreases in transcript and/or protein levels corresponding to two critical genes required for plant Taxol biosynthesis. Taxol showed fungicidal activity against fungal pathogens of conifer wood, the natural habitat of the Taxol-producing endophyte. Consistent with other Taxol-producing endophytes, SSM001 was resistant to Taxol. CONCLUSIONS: These results suggest that the variation in Taxol content between intact Taxus plants and/or tissues is at least in part caused by varying degrees of transcriptional elicitation of plant Taxol biosynthetic genes by its Taxol-producing endophyte. As Taxol is a fungicide, and the endophyte is resistant to Taxol, we discuss how this endophyte strategy may be to prevent colonization by its fungal competitors but at minimal metabolic cost to itself.

Induction of a ricinosomal-protease and programmed cell death in tomato endosperm by gibberellic acid.

Several examples of programmed cell death (PCD) in plants utilize ricinosomes, organelles that appear prior to cell death and store inactive KDEL-tailed cysteine proteinases. Upon cell death, the contents of ricinosomes are released into the cell corpse where the proteinases are activated and proceed to degrade any remaining protein for use in adjacent cells or, in the case of nutritive seed tissues, by the growing seedling. Ricinosomes containing pro-SlCysEP have been observed in anther tissues prior to PCD and ricinosome-like structures have been observed in imbibed seeds within endosperm cells of tomato. The present study confirms that the structures in tomato endosperm cells contain pro-SlCysEP making them bona fide ricinosomes. The relative abundance of pro- versus mature SlCysEP is suggested to be a useful indicator of the degree of PCD that has occurred in tomato endosperm, and is supported by biochemical and structural data. This diagnostic tool is used to demonstrate that a sub-region of the micropylar endosperm surrounding the emerged radical is relatively long-lived and may serve to prevent loss of mobilized reserves from the lateral endosperm. We also demonstrate that GA-induced reserve mobilization, SlCysEP accumulation and processing, and PCD in tomato endosperm are antagonized by ABA.

Hypothesis/review: contribution of putrescine to 4-aminobutyrate (GABA) production in response to abiotic stress.

4-Aminobutyrate (GABA) accumulates in various plant parts, including bulky fruits such as apples, in response to abiotic stress. It is generally believed that the GABA is derived from glutamate, although a contribution from polyamines is possible. Putrescine, but not spermidine and spermine, generally accumulates in response to the genetic manipulation of polyamine biosynthetic enzymes and abiotic stress. However, the GABA levels in stressed plants are influenced by processes other than putrescine availability. It is hypothesized that the catabolism of putrescine to GABA is regulated by a combination of gene-dependent and -independent processes. The expression of several putative diamine oxidase genes is weak, but highly stress-inducible in certain tissues of Arabidopsis. In contrast, candidate genes that encode 4-aminobutyraldehyde dehydrogenase are highly constitutive, but not stress inducible. Changes in O(2) availability and cellular redox balance due to stress may directly influence the activities of diamine oxidase and 4-aminobutyraldehyde dehydrogenase, thereby restricting GABA formation. Apple fruit is known to accumulate GABA under controlled atmosphere storage and therefore could serve as a model system for investigating the relative contribution of putrescine and glutamate to GABA production.

Ricinosomes predict programmed cell death leading to anther dehiscence in tomato.

Successful development and dehiscence of the anther and release of pollen are dependent upon the programmed cell death (PCD) of the tapetum and other sporophytic tissues. Ultrastructural examination of the developing and dehiscing anther of tomato (Solanum lycopersicum) revealed that cells of the interlocular septum, the connective tissue, the middle layer/endothecium, and the epidermal cells surrounding the stomium all exhibit features consistent with progression through PCD. Ricinosomes, a subset of precursor protease vesicles that are unique to some incidents of plant PCD, were also present in all of these cell types. These novel organelles are known to harbor KDEL-tailed cysteine proteinases that act in the final stages of corpse processing following cell death. Indeed, a tomato KDEL-tailed cysteine proteinase, SlCysEP, was identified and its gene was cloned, sequenced, and characterized. SlCysEP transcript and protein were restricted to the anthers of the senescing tomato flower. Present in the interlocular septum and in the epidermal cells surrounding the stomium relatively early in development, SlCysEP accumulates later in the sporophytic tissues surrounding the locules as dehiscence ensues. At the ultrastuctural level, immunogold labeling localized SlCysEP to the ricinosomes within the cells of these tissues, but not in the tapetum. It is suggested that the accumulation of SlCysEP and the appearance of ricinosomes act as very early predictors of cell death in the tomato anther.