Kaempferol rhamnoside catabolism in rosette leaves of senescing Arabidopsis and postharvest stored radish.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-ß-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

Bibenzyl synthesis in Cannabis sativa L.

This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the 'bibenzyls'. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this 'bibenzyl synthase' enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing 'parts prospecting' inventory that supports the rational metabolic engineering of natural product therapeutics.

The Induction of the Isoflavone Biosynthesis Pathway Is Associated with Resistance to Common Bacterial Blight in Phaseolus vulgaris L.

Xanthomonas axonopodis infects common bean (Phaseolus vulgaris L.) causing the disease common bacterial blight (CBB). The aim of this study was to investigate the molecular and metabolic mechanisms underlying CBB resistance in P. vulgaris. Trifoliate leaves of plants of a CBB-resistant P. vulgaris recombinant inbred line (RIL) and a CBB-susceptible RIL were inoculated with X. axonopodis or water (mock treatment). Leaves sampled at defined intervals over a 48-h post-inoculation (PI) period were monitored for alterations in global transcript profiles. A total of 800 genes were differentially expressed between pathogen and mock treatments across both RILs; approximately half were differentially expressed in the CBB-resistant RIL at 48 h PI. Notably, there was a 4- to 32-fold increased transcript abundance for isoflavone biosynthesis genes, including several isoflavone synthases, isoflavone 2'-hydroxylases and isoflavone reductases. Ultra-high performance liquid chromatography-tandem mass spectrometry assessed leaf metabolite levels as a function of the PI period. The concentrations of the isoflavones daidzein and genistein and related metabolites coumestrol and phaseollinisoflavan were increased in CBB-resistant RIL plant leaves after exposure to the pathogen. Isoflavone pathway transcripts and metabolite profiles were unaffected in the CBB-susceptible RIL. Thus, induction of the isoflavone pathway is associated with CBB-resistance in P. vulgaris.

In through the out door: Biochemical mechanisms affecting flavonoid glycoside catabolism in plants.

Plants are the sole source of flavonoids, a chemical category that includes flavonols. For the most part, flavonols occur as glycosides with numerous postulated biological roles in plants, including photoprotection, modulation of hormone translocation, and sequestration of reactive oxygen species. Flavonol glycosides are often considered as dead-end metabolites because related flavonoids (i.e., anthocyanins) occur in terminal tissues such as flowers and fruit, but recent evidence points to their turnover in planta, including developing photosynthetic tissues. Although microbial degradation pathways for flavonol glycosides of plant origin are well described, plant catabolic pathways are little studied by comparison. This review will address our current understanding of biochemical processes leading to the loss of flavonol glycosides in plants, with a specific emphasis on the evidence for flavonol-specific ß-glucosidases. Complete elucidation of these catabolic pathways is dependent on the identification of regiospecific modifying steps, including enzymes associated with the hydrolysis of rhamnosylated flavonols, as well as flavonol peroxidation and their encoding genes. Herein, we highlight challenges for the identification of hypothetical plant α-rhamnosidases and peroxidases involved in flavonol glycoside degradation, and the potential biological role of this catabolism in mitigating oxidative stress in developing and postharvest plant tissues.

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.

Metabolic Alterations in Postharvest Pear Fruit As Influenced by 1-Methylcyclopropene and Controlled Atmosphere Storage.

This study assessed the impact of 1-methylcyclopropene (1-MCP) and controlled atmosphere (CA) on the metabolism of targeted amino acids, organic acids, and antioxidants in stored 'AC Harrow Crisp' pears and their relationships to storage disorders. Pears were treated with 0 or 300 nL L-1 1-MCP and stored at 0 °C under ambient air or CA. Spectrophotometric assays demonstrated that glutathione levels fluctuated with storage and were most preserved by 1-MCP under ambient air. HPLC analysis revealed that ascorbate concentrations declined with storage and were little affected by 1-MCP and CA. Citrate, lactate, and fumarate accumulated with storage but were differentially affected by 1-MCP. Aspartate and glutamate concentrations were greater with 1-MCP; γ-aminobutyrate accumulated in disordered fruit. Principal component analysis demonstrated that alterations in citrate and fumarate were, respectively, correlated with internal breakdown and senescent scald. γ-Aminobutyrate and alanine were associated with internal cavities. All disorders were associated with antioxidant depletion.

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.

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.

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

BACKGROUND: Edible 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. RESULTS: Seed 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. CONCLUSION: Proanthocyanidin 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.

An Apoplastic ß-Glucosidase is Essential for the Degradation of Flavonol 3-O-ß-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis ß-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-ß-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-ß-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-ß-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and quercetin 3-O-ß-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-ß-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus, BGLU15 is essential for catabolism of flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides in Arabidopsis.

Changes in light quality alter physiological responses of soybean to thiamethoxam.

MAIN CONCLUSION: The interaction between neighboring weed-induced far-red enriched light and thiamethoxam can significantly alter soybean seedling morphology, nodulation, isoflavone levels, UV-absorbing phenolics, and carbon and nitrogen content. Neonicotinoid insecticides that are widely used on major crop plants can enhance plant growth and yield. Although the underlying mechanism of this enhanced growth and yield is not clear, recent studies suggest that neonicotinoids such as thiamethoxam (TMX) may exert their effects at least in part via signals that involve salicylic acid (SA) and jasmonic acid (JA). In the current research, effects of TMX on morphological and physiological responses of soybean have been compared under far-red-depleted (FR-D) and far-red-enriched (FR-E) light reflected by neighboring weeds. TMX significantly enhanced shoot and root growth but did not prevent stem elongation under FR-E light. Also, TMX did not prevent reductions in shoot carbon content and shoot carbon to nitrogen ratio under FR-E light. Despite similarities between these TMX effects in soybean and those known for SA and JA in other plant species, TMX significantly enhanced root-nodule numbers per plant and levels of root isoflavones malonyl-daidzin and malonyl-genistin under FR-E light only. These results suggest that the combined effect of FR-E light and TMX triggers a mechanism that operates concomitantly to enhance root isoflavones and nodulation in soybean.

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 ß-glucosidase BGLU15 attacks flavonol 3-O-ß-glucoside-7-O-α-rhamnosides.

Kaempferol and quercetin 3-O-ß-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 ß-glucosidase (BGLU) action. Evidence for flavonol 3-O-ß-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-ß-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-ß-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 ß-glucosides showed that recombinant BGLU15 preferentially hydrolyses the 3-O-ß-glucosides of flavonols, but does not attack quercetin 3-O-α-rhamnoside, quercetin 3-O-ß-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-ß-glucoside-7-O-α-rhamnosides in Arabidopsis is dependent upon the enzyme-mediated cleavage of the 3-O-ß linked glucose moiety.

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.

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.

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.

Metabolism of the folate precursor p-aminobenzoate in plants: glucose ester formation and vacuolar storage.

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K(m) for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k(cat)/K(m) value than the others and a far lower apparent K(m) for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [(14)C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The K(eq) for the pABA esterification reaction was found to be 3 x 10(-3). Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this K(eq) value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [(14)C]pABA-fed pea leaves were estimated to contain> or =88% of the [(14)C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [(3)H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.