Gale G. Bozzo

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.

Reappraisal of nitrogen use efficiency in rice overexpressing glutamine synthetase1

Cytosolic glutamine synthetase (GS1) is responsible for the primary assimilation of ammonia, and a role in nitrogen (N) remobilization is implicated from its vascular localization and enhanced expression during senescence. This paper tested the hypothesis that overexpression (OX) of GS1 in rice improves utilization N use efficiency (UtE = spikelet yield/shoot N content). Three GS1 OX lines were identified using activity assays and quantitative polymerase chain reaction. Physiological analysis of the OX lines, as well as azygous and wild-type (Wt) controls, was conducted with mature plants after growth under varying nitrate conditions (non-limiting N, limiting N, transfer from non-limiting N to limiting N at panicle emergence) and growth environments (growth chamber vs greenhouse). Overall, OX lines did not differ from azygous controls in vegetative yield or shoot N content. In two of the three growth trials (i.e. the growth chamber trials) harvest index, N harvest index (spikelet N content/shoot N content) and UtE were generally enhanced in the OX lines relative to their azygous controls. These characteristics were highly correlated with percent spikelets filled and spikelet number. Thus, N partitioning in rice during grain filling could be altered by GS1 OX, resulting in improved UtE. Unfortunately, GS OX did not result in more efficient use of N under limiting N than under non-limiting N, and is therefore unlikely to result in the use of less N under field conditions. Transformation effects significantly hindered the productivity of the OX lines, but backcrossing to the Wt should overcome this.

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

BackgroundThe 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 Ca2+/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity.ResultsGAD activity in cell-free extracts of apple fruit was stimulated by Ca2+/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 Ca2+/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 Ca2+/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.ConclusionsPlant 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.

Biocatalytic Synthesis of Quercetin 3‐O‐Glucoside‐7‐O‐Rhamnoside by Metabolic Engineering of Escherichia coli

Flavonol glycosides, like quercetin 3-O-glucoside (1) and the bisglycoside quercetin-3-O-glucoside-7-O-rhamnoside (2) are plant natural products exhibiting numerous biological activities. 2] Compound 2 is rare, but has been described for Capsicum species and Arabidopsis thaliana, and is often found in complex mixtures with other flavonols. 3–5] Hence, purification from plants is not practical. Here, we report the regioselective synthesis and purification of 2 from an Escherichia coli expression strain harbouring a rhamnose synthase and a flavonol 7O-rhamnosyltransferase from Arabidopsis thaliana. Approximately 350 quercetin derivatives exist, they all come from plants. In nature, quercetin occurs as glycosides with pentoses or hexoses (e.g. , rhamnose) conjugated to any of five available hydroxy groups. 8] Glycosylation of flavonols increases their aqueous solubility relative to their aglycones, hence enhancing the absorption of quercetin from the small intestine in humans. In A. thaliana, 11 quercetin glycosides are known, including 2 and others distinguished by the regiospecific attachment of one or two sugar moieties at the 3-O and/or 7-O positions. Flavonol bisglycoside production is catalysed by uridine diphosphate (UDP)-dependent glycosyltransferase (UGT). In Arabidopsis, 2 is derived from 1 by a UDP–rhamnose-dependent flavonol 7-O-rhamnosyltransferase (AtUGT89C1). 11] UDP– rhamnose is derived from UDP–glucose by the activity of rhamnose synthase (RHM). Ablation of AtRHM1 culminates in a marked reduction in flavonol 3-O-glucoside-7-O-rhamnoside levels in Arabidopsis leaves and flowers, thus pointing to a sequence of steps in the regioselective synthesis of 2 in plants. Bacteria are rich in nucleotide sugars and UGTs, but lack flavonols. Moreover, bacteria contain UDP–glucose and thymine diphosphate rhamnose (TDP–rhamnose); however, UDP–rhamnose is absent. Quercetin fed to E. coli expressing AtUGT89C1 together with a flavonol 3-O-glucosyltransferase (AtUGT78D2) yields a complex mixture consisting of compounds 1 and 2 together with quercetin 3-O-glucoside-7-Oglucoside, quercetin 3-O-(N-acetyl)glucosamine and quercetin 3-O-(N-acetyl)glucosamine-7-O-rhamnoside. In the absence of UDP–rhamnose, the aforementioned transformants use U(T)DP–glucose and UDP–N-acetylglucosamine in addition to TDP–rhamnose to modify 1 leading to by-products. Alternatively, dual expression of plant UGT and RHM genes in E. coli generates rhamnosylated quercetin, but this has not been attempted for the rare bisglycoside 2. To limit the number of by-products produced during biocatalysis the synthesis of 2 may be facilitated by feeding b-glucoside 1 to metabolically engineered E. coli. b-Glucosides are actively absorbed by E. coli, but are not metabolised by wildtype strains; this eliminates any possibility of their hydrolysis in a biocatalytic reaction. We examined the feasibility of feeding 1 to an E. coli expression strain containing two plasmids, one of which carried AtUGT89C1 and the other AtRHM1 (Scheme 1). Moreover, we describe a simple chromatography procedure for purifying this bisglycoside. To determine whether regioselective synthesis of 2 was possible, pET41b and pET32b expression vectors harbouring AtUGT89C1 and AtRHM1, respectively, were co-transformed into E. coli BL21(DE3). Compound 1 (1 mg per 50 mL culture) was added to culture media of E. coli dually expressing AtUGT89C1 and AtRHM1, E. coli harbouring one of these plant genes together with an empty plasmid, or cells transformed with two empty plasmids. At the end of the biocatalysis reaction, quercetin conjugates from the spent culture media and cell lysates of AtRHM1/AtUGT89C1 transformants were extracted and analysed. As much as 40 % of 1 was recovered from cell lysates, regardless of whether a product was detected (Figure 1 A); however, it is possible that part of the absorbed compound 1 was refluxed to the medium, as previous studies have shown that 60–80 % of flavonol aglycone and monoglycoside are secreted during biocatalysis. HPLC-DAD (DAD = diode array detector) revealed a single product with a retention time (tR = 6.1 min) distinct from that of 1 (tR = 8.0 min; Figure 1). The product’s absorption maxima (256, 354 nm, in 20 % acetonitrile containing 0.1 % formic acid; Supporting Information) were comparable to those of compound 2 isolated from whole-plant Arabidopsis. Reaction product levels were 1.5 times greater in E. coli AtRHM1/AtUGT89C1 transformants than in cells expressing only AtUGT89C1 (Supporting Information). A product in E. coli expressing only AtUGT89C1 (Supporting Information) suggests rhamnosylation of compound 1 is supplemented by endogenous TDP–rhamnose. As expected, no product was formed in cells expressing only AtRHM1 or those harbouring two empty plasmids (Supporting Information), thus pointing to a lack of natural flavonol bisglycoside biosynthesis in bacteria. The majority of the reaction product (80 %) was present in cells, with the remainder in the spent culture medium (Figure 1 B). Although the proportion of 2 recovered in the spent cell culture medium was lower than in the cellular fraction, this phenomenon is consistent with the partial secretion of quercetin conjugates from E. coli flavonol UGT transformants. A previous report found 2 localised primarily to the E. coli culture medium; this might be a consequence of a longer incubation period and/or cultivation at 70 % higher cell density under [a] J. Roepke, Dr. G. G. Bozzo Department of Plant Agriculture, University of Guelph 50 Stone Road E. , Guelph, ON, N1G 2W1 (Canada) E-mail : gbozzo@uoguelph.ca Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201300474.

Impact of 1-methylcyclopropene and controlled atmosphere storage on polyamine and 4-aminobutyrate levels in “Empire” apple fruit

1-Methylcyclopropene (1-MCP) delays ethylene-meditated ripening of apple (Malus domestica Borkh.) fruit during controlled atmosphere (CA) storage. Here, we tested the hypothesis that 1-MCP and CA storage enhances the levels of polyamines (PAs) and 4-aminobutyrate (GABA) in apple fruit. A 46-week experiment was conducted with “Empire” apple using a split-plot design with four treatment replicates and 3°C, 2.5 kPa O2, and 0.03 or 2.5 kPa CO2 with or without 1 μL L-1 1-MCP. Total PA levels were not elevated by the 1-MCP treatment. Examination of the individual PAs revealed that: (i) total putrescine levels tended to be lower with 1-MCP regardless of the CO2 level, and while this was mostly at the expense of free putrescine, large transient increases in soluble conjugated putrescine were also evident; (ii) total spermidine levels tended to be lower with 1-MCP, particularly at 2.5 kPa CO2, and this was mostly at the expense of soluble conjugated spermidine; (iii) total spermine levels at 2.5 kPa CO2 tended to be lower with 1-MCP, and this was mostly at the expense of both soluble and insoluble conjugated spermine; and (iv) total spermidine and spermine levels at 0.03 kPa were relatively unaffected, compared to 2.5 kPa CO2, but transient increases in free spermidine and spermine were evident. These findings might be due to changes in the conversion of putrescine into higher PAs and the interconversion of free and conjugated forms in apple fruit, rather than altered S-adenosylmethionine availability. Regardless of 1-MCP and CO2 treatments, the availability of glutamate showed a transient peak initially, probably due to protein degradation, and this was followed by a steady decline over the remainder of the storage period which coincided with linear accumulation of GABA. This pattern has been attributed to the stimulation of glutamate decarboxylase activity and inhibition of GABA catabolism, rather than a contribution of PAs to GABA production.

Oxidative metabolism is associated with physiological disorders in fruits stored under multiple environmental stresses

In combination with low temperature, controlled atmosphere storage and 1-methylcyclopropene (ethylene antagonist) application are used to delay senescence of many fruits and vegetables. Controlled atmosphere consists of low O2 and elevated CO2. When sub-optimal partial pressures are used, these practices represent multiple abiotic stresses that can promote the development of physiological disorders in pome fruit, including flesh browning and cavities, although there is some evidence for genetic differences in susceptibility. In the absence of surface disorders, fruit with flesh injuries are not easily distinguished from asymptomatic fruit until these are consumed. Oxidative stress metabolites tend to accumulate (e.g., γ-aminobutyrate) or rapidly decline (e.g., ascorbate and glutathione) in vegetative tissues exposed to hypoxic and/or elevated CO2 environments. Moreover, these phenomena can be associated with altered energy and redox status. Biochemical investigations of Arabidopsis and tomato plants with genetically-altered levels of enzymes associated with the γ-aminobutyrate shunt and the ascorbate-glutathione pathway indicate that these metabolic processes are functionally related and critical for dampening the oxidative burst in vegetative and fruit tissues, respectively. Here, we hypothesize that γ-aminobutyrate accumulation, as well energy and antioxidant depletion are associated with the development of physiological injury in pome fruit under multiple environmental stresses. An improved understanding of this relationship could assist in maintaining the quality of stored fruit.

Arabidopsis thaliana β-glucosidase BGLU15 attacks flavonol 3-O-β-glucoside-7-O-α-rhamnosides

Kaempferol and quercetin 3-O-β-glucoside-7-O-α-rhamnoside (K3G7R and Q3G7R, respectively) are major flavonol bisglycosides accumulating in Arabidopsis thaliana with synergistic abiotic stresses (i.e., nitrogen deficiency and low temperature, NDLT). However, these molecules disappear rapidly during recovery from NDLT. Typically, catabolism of related chemicals relies on β-glucosidase (BGLU) action. Evidence for flavonol 3-O-β-glucoside-7-O-α-rhamnoside BGLU activity is provided here. Major losses of Q3G7R and K3G7R coincided with an approximate 250% induction in flavonol 3-O-β-glucoside-7-O-α-rhamnoside BGLU activity within 2days of NDLT recovery relative to plants cultured under nitrogen sufficiency and high temperature (NSHT, control). QTOF-MS/MS established the product of Q3G7R hydrolysis in the presence of Arabidopsis cell free extracts was quercetin 7-O-α-rhamnoside. A phylogenetic analysis of the Arabidopsis glycoside hydrolase family 1 identified BGLU15 (At2g44450) and five other members that cluster with Fabaceae hydrolases known to attack isoflavones and isoflavonoids, which are structurally somewhat related to flavonol 3-O-β-glucoside-7-O-α-rhamnosides. Real time quantitative PCR analysis established a 300% higher expression of BGLU15 within 1day of the recovery from NDLT relative to control plants; lower or negligible changes in expression were evident for the remaining BGLUs. Recombinant thioredoxin-His6-tagged mature BGLU15 protein was expressed in Escherichia coli and purified to homogeneity. A comparison of a wide spectrum of β-glucosides showed that recombinant BGLU15 preferentially hydrolyses the 3-O-β-glucosides of flavonols, but does not attack quercetin 3-O-α-rhamnoside, quercetin 3-O-β-galactoside and rutin. BGLU15 displayed the highest catalytic efficiency for Q3G7R and K3G7R yielding their respective 7-O-rhamnosides as products; flavonol 3-O-glucosides were also attacked, albeit with lower efficiency. Together, it appears the loss of flavonol 3-O-β-glucoside-7-O-α-rhamnosides in Arabidopsis is dependent upon the enzyme-mediated cleavage of the 3-O-β linked glucose moiety.

Proanthocyanidin accumulation and transcriptional responses in the seed coat of cranberry beans (Phaseolus vulgaris L.) with different susceptibility to postharvest darkening

BackgroundEdible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown.ResultsSeed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products.ConclusionProanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.

Effects of elevated CO2 and 1-methylcyclopropene on storage-related disorders of Ontario-grown Empire apples

Deyman, K. L., Chiu, G., Liu, J., Brikis, C. J., Trobacher, C. P., DeEll, J. R., Shelp, B. J. and Bozzo, G. G. 2014. Effects of elevated CO2 and 1-methylcyclopropene on storage-related disorders of Ontario-grown Empire apples. Can. J. Plant Sci. 94: 857-865. The impact of 1-methylcyclopropene (1-MCP) application on CO2-induced physiological injury in Empire apple fruit during controlled atmosphere storage was assessed over a 3-yr period using an experimental design involving multiple treatment replicates. Fruit harvested at optimal maturity from one or two orchards were treated with or without 1 µL L-1 1-MCP, then chilled at 0 or 3°C under various CO2 partial pressures (5, 2.5 or 0.03 kPa CO2) in the presence of 2.5 kPa O2 for up to 46 wk using a split-plot design. Fruit were sampled periodically for assessment of flesh browning and external peel injury. The maximal incidence of external CO2 injury varied from 15 to 100% over the 3 yr, and the most rapid development of this disorder was evident at 5 kPa CO2. The incidence of external CO2 injury as a function of storage time was influenced by orchard location and storage temperature. Moreover, the incidence of flesh browning at 0°C and 5 kPa CO2 was influenced slightly by orchard; this disorder was never higher than 30%, and the impact of elevated CO2 was inconsistent across years. Notably, there was no evidence for negative effects of 1-MCP on the incidence of storage-related disorders.

Plant Glyoxylate/Succinic Semialdehyde Reductases: Comparative Biochemical Properties, Function during Chilling Stress, and Subcellular Localization

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.