[Show abstract][Hide abstract] ABSTRACT: Four breeding lines (BLs) of white clover, which range in terms of their disposition to increase biomass in response to low or high phosphate (P) levels in soil, were evaluated further when grown hydroponically. BL 45 increased both fresh and dry weight in response to P-deprivation, while BL 49 displayed the same significant stimulation in response to sufficient P. However, when compared these lines did not accumulate any significantly higher levels of P, and all four lines showed the same changes to root:shoot ratio in response to P-deprivation, as well as the induction of both soluble and cell-wall-associated acid phosphatase activity. These results confirm that there is no direct relationship between growth (as an increase in biomass) and acid phosphatase activity and further some responses to P-deprivation may be common to all genetic backgrounds suggesting that they are not part of the intricate mechanisms governing these responses.
Journal of Plant Nutrition 07/2014; 37(9). · 0.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The consideration as to how plants uptake and transport phosphorus (P) is of significant agronomic and economic importance, in part driven by finite reserves of rock phosphate. Our understanding of these mechanisms has been greatly advanced, particularly with respect to the responses of plants to P deficiency and the genetic dissection of the signalling involved. Further, the realization that there are two tiers of transcriptional responses, the local, in which inorganic P (Pi) acts as an external signal independent of the endogenous P level, and the systemic involving root–shoot signalling, has now added a dimension of both clarity and complexity. Notwithstanding, it is now clear that the hormone ethylene plays a key role in mediating both levels of responses. This review, therefore, covers the role of ethylene in terms of mediating responses to P deficiency. The evidence that Pi supply regulates ethylene biosynthesis and sensitivity, and that this, in turn, regulates changes in root system architecture and in Pi-deprivation responses is examined here. While ethylene is the focus, the key interactions with auxin are also assessed, but interactions with the other hormone groups, which have recently been reviewed, are not covered. The emerging view that ethylene is a multi-faceted hormone in terms of mediating responses to P deficiency invites the dissection of the transcriptional cues that mediate changes in ethylene biosynthesis and/or sensitivity. Knowledge of the nature of such cues will subsequently reveal more of the underpinning interactions that govern P responses and provide avenues for the production of germplasm with an improved phosphate use efficiency.
[Show abstract][Hide abstract] ABSTRACT: Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterized with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion has a single functional SiR gene and also expresses an unprocessed pseudogene (φ-SiR). Northern and qPCR analysis revealed differences in expression pattern between the SiR gene and the pseudogene. Western analysis using antibodies raised to a recombinant SiR revealed that the enzyme is present in chloroplasts and phylogenetic analysis has shown that the onion protein groups with lower eudicots. In hydroponically-grown plants, levels of SiR transcripts were significantly higher in the roots of S-sufficient when compared with S-deficient plants of the pungent cultivar 'W202A' but not the less pungent cultivar 'Texas Grano'. In these same treatments, a higher level of enzyme activity was observed in the S-sufficient treatment in leaves of both cultivars before and after bulbing. In a factorial field trial with and without sulfur fertilization, a statistically significant increase in SiR activity was observed in the leaves of the pungent cultivar 'Kojak' in response to added S but not in the less pungent cultivar 'Encore'.
[Show abstract][Hide abstract] ABSTRACT: Background and aims Aluminium (Al) toxicity limits pasture production in acidic soils, and there is evidence that antioxidant enzymes such as superoxide dismutase (SOD) play a key role against Al-induced oxidative stress. We have investigated (i) the relation-ship between Al-toxicity and oxidative damage as well as the time-course of SOD activity and (ii) the expression profiles of two SOD isoforms in the roots of two ryegrass cultivars. Methods Jumbo (Al-sensitive) and Nui (Al-semi-toler-ant) ryegrass cultivars were cultured hydroponically with 0 or 0.2 mM Al. Roots were colleted during 48 h to determine Al uptake, lipid peroxidation, SOD activity and the expression of LpCu/Zn-SOD and LpFe-SOD isolated genes. Results LpCu/Zn-SOD and LpFe-SOD were classified phylogenetically as putative mitochondrial and plastidial SODs, respectively. Although Al accumulation did not differ between the two cultivars, lipid peroxidation increased 2.2-fold in Jumbo but only scarcely in Nui by the end of the time-course. An early response in terms of total SOD activity and differential expression of LpCu/Zn-SOD and LpFe-SOD genes occurred in both cultivars after Al treatment. Conclusions SOD isoforms were up-regulated over a longer period in Nui compared with Jumbo, leading finally to a greater protection in roots against the short term Al-toxicity. Keywords Aluminium . Cu/Zn-SOD . Fe-SOD . Gene expression . TBARS . Ryegrass
Plant and Soil 01/2012; 350:353. · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine the effect on Mn treatment on the ACO gene family of white clover [Trifolium repens (L.) cv. Grasslands Challenge], rooted stolon cuttings were maintained in modified Hoaglands medium, at pH 5.4, containing either 5.2 μM Mn or 100 μM Mn over a 9-day time course. A significant uptake of Mn was observed in leaf tissue of plants grown in the 100 μM Mn treatment after 24 h and the content increased in these plants to reach 334 mg/kg DW at the conclusion of the time course. The growth of plants, measured as the petiole extension rate (PER), was significantly less in the 100 μM Mn treatment by day 9, while significantly less accumulation of leaf biomass was observed by day 7. The activity of a cell wall-associated H(2)O(2)-generating NADH peroxidase was shown to be higher in the 100 μM Mn treatment after day 5 of the time course while no significant difference in a H(2)O(2)-consuming guaiacol peroxidase activity was observed between the two Mn treatments. The expression of two leaf-associated ACC oxidase (ACO) genes, TR-ACO2 and TR-ACO3 was examined over the 9-day course but no difference between the two treatments was observed. In contrast, TR-ACO2 enzyme activity was measured and shown to decrease in the 100 μM Mn treatment after day 5 of the time course, with a concomitant decrease in TR-ACO2 accumulation, as determined by western analysis. Using 2DE and western analysis, evidence for post-translational modification of TR-ACO2 was observed.
Plant Physiology and Biochemistry 04/2011; 49(9):1013-9. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the regulation of sulphur (S)-assimilation in onion further at the biochemical level, the pungent cultivar W202A and the milder cultivar Texas Grano 438 PVP (TG) have been grown in S-sufficient (S(+); 4meqS(-1)) or S-deficient (S(-); 0.1meqS(-1)) growth conditions, and tissues excised at the seedling stage (pre-bulbing; ca. 10-weeks-old) and at the mature stage (bulbing; ca. 16-weeks-old). S-supply negatively influenced adenosine-5'-phosphosulphate (APS) reductase (APR) enzyme activity in both cultivars at bulbing only, and a higher abundance of APR was observed in both cultivars at bulbing in response to low S-supply. In contrast, S-supply significantly influenced ATP sulphurylase (ATPS) activity in leaf tissues of W202A only, and only at bulbing, while an increase in abundance in response to high S-supply was observed for both cultivars at bulbing. To investigate the regulation of the ATPS enzyme activity and accumulation further, activity was shown to decrease significantly in roots at bulbing in the S-deficient treatment in both cultivars, a difference that was only supported by western analyses in W202A. Phylogenetic analysis revealed that AcATPS1 groups in a broad monocot clade with the closest sequences identified in Sorghum bicolour, Zea mays and Oryza sativa, but with some support for a divergence of AcATPS1. Detection of ATPS in leaf extracts after two dimensional gel electrophoresis (2-DE) revealed that the protein may undergo post-translational modification with a differential pattern of ATPS accumulation detected in both cultivars over the developmental progression from the seedling to the bulbing stage. Treatment of leaf extracts of W202A to dephosphorylate proteins resulted in the loss of immuno-recognised ATPS spots after 2-DE separation, although enzyme activity was not influenced. These results are discussed in terms of the tiers of control that operate at the biochemical level in the reductive S-assimilation pathway in a S-accumulating species particularly during the high-S-demanding bulbing stage.
[Show abstract][Hide abstract] ABSTRACT: Organosulfur compounds are major sinks for assimilated sulfate in onion (Allium cepa L.) and accumulation varies widely due to plant genotype and sulfur nutrition. In order to better characterise sulfur metabolism phenotypes and identify potential control points we compared plant composition and transcript accumulation of the primary sulfur assimilation pathway in the high pungency genotype 'W202A' and the low pungency genotype 'Texas Grano 438' grown hydroponically under S deficient (S-) and S-sufficient (S+) conditions. Accumulation of total S and alk(en)yl cysteine sulfoxide flavour precursors was significantly higher under S+ conditions and in 'W202A' in agreement with previous studies. Leaf sulfate and cysteine levels were significantly higher in 'W202A' and under S+. Glutathione levels were reduced by S- treatment but were not affected by genotype, suggesting that thiol pool sizes are regulated differently in mild and pungent onions. The only significant treatment effect observed on transcript accumulation in leaves was an elevated accumulation of O-acetyl serine thiol-lyase under S-. By contrast, transcript accumulation of all genes in roots was influenced by one or more treatments. APS reductase transcript level was not affected by genotype but was strongly increased by S-. Significant genotype×S treatment effects were observed in a root high affinity-sulfur transporter and ferredoxin-sulfite reductase. ATP sulfurylase transcript levels were significantly higher under S+ and in 'W202A'.
[Show abstract][Hide abstract] ABSTRACT: Protein extracts, made to leaves harvested from the stolons of the pasture legume white clover (Trifolium repens L.) at two developmental stages (newly initiated; onset of senescence) were purified further using reverse-phase and ionexchange
chromatography. Fractions enriched with the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase were
selected for each stage and the final, partially purified fraction was subjected to twodimensional gel electrophoresis (2DE).
Antibodies raised against a recombinant ACC oxidase (ACO) from white clover (antiTR-ACO2) recognised a series of spots of
differing pI suggesting that ACO undergoes post-translational modifications. Further, the pattern differed between the ACO
proteins partially purified from newly initiated leaves with leaves at the onset of senescence suggesting that the environmental
and developmental cues that operate in each tissue influences the type and/or degree of post-translational modifications of
the ACO protein. MALDI-TOF mass spectrometry was used to identify protein spots associated with the ACO proteins. Protein
with identities to an ACO isoform from Oryza sativa, and a phosphoribulokinase from Arabidopsis thaliana were identified in the 2DE separations from newly initiated leaves, while an isoflavone reductase from Medicago sativa was identified in the 2DE separation of the senescent leaf extract.
Additional key wordsACC oxidase–ethylene synthesis–isoflavone reductase–leaf development–phosphoribulokinase
Biologia Plantarum 01/2011; 55(2):261-268. · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification and expression of two ACC oxidase (ACO) genes during leaf development in Trifolium occidentale (L.), one of the putative ancestral genomes of the allotetraploid, T. repens (L.; white clover), is described. In common with observations made in T. repens, the ACO genes displayed differential expression, with a TR-ACO2-like gene (designated TO-ACO2) confined to developing and early mature-green leaf tissue while expression of a TR-ACO3-like gene (designated TO-ACO3) is highest in leaves at the onset and during senescence. Biochemical analysis of TO-ACO2 revealed that both accumulation of the protein (determined by western analysis with a TR-ACO2 antibody) and enzyme activity matched the transcriptional activity of TO-ACO2. Western analysis also revealed that the Tr-ACO2 antibody recognised a protein of 37 kDa as a putative TP-ACO2 in T. pallescens. The 3'-UTRs of TO-ACO2 and TO-ACO3 were then compared with the 3'-UTRs of a TR-ACO2-like and TR-ACO3-like gene from T. pallescens, the other proposed ancestral genome (or closely related to the ancestor) of T. repens, with identity values of 87.8% for the ACO2-like genes and 94.8% for the ACO3-like genes. Comparison of the 3'-UTRs of TO-ACO2 with a TO-ACO2-like gene in T. repens (designated TR(O)-ACO2) and TP-ACO2 with a TP-ACO2-like gene in T. repens (designated TR(P)-ACO2) revealed identities of 100% and 96.6%, respectively, lending good support to T. occidentale as one of the ancestral genomes of T. repens. A similar comparison of the 3'-UTRs of TO-ACO3 with a TO-ACO3-like gene in T. repens (designated TR(O)-ACO3) and TP-ACO3 with a TP-ACO3-like gene in T. repens (designated TR(P)-ACO3) revealed identities of 99.5% and 97.9%, respectively, again supporting T. occidentale as one of the ancestral genomes. Further, these data confirm that both TO-ACO-like and TP-ACO-like genes are expressed in the allotetraploid T. repens.
Plant Physiology and Biochemistry 01/2011; 49(4):420-6. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The regulation of proline biosynthesis has been examined in callus and cell cultures of the indica-type rice cultivar Khao
Dawk Mali 105 (KDML105) in response to a saline treatment (250mM NaCl) in terms of the expression of Δ1-pyrroline-5-carboxylate reductase (OsP5CR) and members of the gene family encoding the rate-determining enzyme, Δ1-pyrroline-5-carboxylate synthase (designated OsP5CS1 and OsP5CS2). Using friable callus, growth was retarded by treatment with 250mM NaCl within 4days, with a significant increase in the
expression of OsP5CS2 by 24h, and a less marked induction in OsP5CS1 and OsP5CR over the same time-course. Cell suspension cultures derived from the friable callus were also treated with 250mM NaCl and
an induction in OsP5CS2 was again observed, although this was not as marked as in the friable callus, and there was no significant change in OsP5CS1 and OsP5CR expression. This is the first report that details the expression of OsP5CS1 and OsP5CS2 in tissue culture and the results show that, in common with whole plants, OsP5CS2 displays a primacy of response to saline treatment. However, this response may require a community of communicating cells,
as occurs in callus tissue, rather than cell suspension cultures. This difference has implications both in terms of the biology
of signaling in response to increased salinity and in the use of tissue culture to screen for saline-tolerant germplasm.
Oryza sativa (L.)-Khao Dawk Mali 105 (KDML105)-Proline accumulation-Proline biosynthesis-Callus culture-Salt treatment
Plant Cell Tissue and Organ Culture 01/2010; 103(3):369-376. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The expression of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (E.C. 1.4.3)(ACO) genes, TR-ACO2 and TR-ACO3 from white clover has been examined in leaf tissue in response to physiological stimuli. In detached mature-green leaves,
the expression of TR-ACO3 (a leaf-senescence-associated ACC oxidase) is induced over a 6h time-course, while expression of TR-ACO2 (expressed constitutively in mature-green leaf tissue) decreased over the same time-course. An almost identical pattern of
expression was observed with detached mature-green leaves treated with ethylene, while wounding also did not influence the
timing of TR-ACO3 induction, significantly. No increase in overall ACO enzyme activity was detectable in crude extracts from wounded and detached
leaf tissue, but after separation of these extracts using hydrophobic interaction column chromatography, two ACO isoforms
were identified in non-wounded mature-green leaves (designated MGI and MGII), and the specific activity of one of these (MGII)
increased in wounded tissues. In attached leaf tissue, TR-ACO2, but not TR-ACO3 expression was induced by treatment with ethylene; an induction that was not observed with prior treatment of plants with
1-MCP. This induction in response to ethylene was also observed at the translational level using antibodies to TR-ACO2. Analysis,
in silico, of the 5′ flanking sequences to identify putative transcriptional binding domains reveals that both the TR-ACO2 and TR-ACO3 5′ flanking sequence contain domains that are associated with an ethylene-response. The significance of these observations
in terms of how the expression of these ACO genes is regulated during leaf ontogeny in vivo is discussed.
Trifolium repens L.-ACC oxidase-Ageing-Ethylene-Leaf development
[Show abstract][Hide abstract] ABSTRACT: Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990bp) and MD-ACO3 (966bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3' untranslated regions (3'-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3'-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K(m)) of 89.39 microM (MD-ACO1), 401.03 microM (MD-ACO2) and 244.5 microM (MD-ACO3) for the substrate ACC, catalytic constants (K(cat)) of 6.6x10(-2) (MD-ACO1), 3.44x10(-2) (Md-ACO2) and 9.14x10(-2) (MD-ACO3) and K(cat)/K(m) (microMs(-1)) values of 7.38x10(-4) microMs(-1) (MD-ACO1), 0.86x10(-4)Ms(-1) (MD-ACO2) and 3.8x10(-4) microMs(-1) (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.
[Show abstract][Hide abstract] ABSTRACT: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra?
Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined.
The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment.
The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.
Annals of Botany 02/2008; 101(2):285-92. · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While it is generally accepted that Neotyphodium lolii and related epichloë endophytes are mutualists that provide important benefits to their perennial ryegrass (Lolium perenne) host plants under conditions of biotic stress, relatively little is known about the effect of endophyte on the host under conditions of abiotic stress. Using genetically identical endophyte infected (E+) and uninfected (E−) clones of perennial ryegrass from a natural and a synthetic association grown under conditions of water stress in a controlled environment, we show that N. lolii had minor effects on morphological responses (leaf elongation rate and ground biomass production) but had more pronounced effects on physiological responses (water use efficiency, relative water content and osmotic potential) by the host. The effects were most marked in the natural association. While levels of proline increased in response to water stress, the presence of endophyte had no effect on those levels. The effect of water stress on endophyte bioprotective metabolites was also examined. Ergovaline levels in pseudostem tissue increased in response to increasing water stress for both N. lolii/L. perenne associations but lolitrem B levels only increased in the natural association. No differences in steady state levels of transcripts from genes known to be required for the synthesis of these alkaloids were observed in response to water stress. This study demonstrated that N. lolii can confer protection to perennial ryegrass from water stress and that levels of the bioprotective metabolites, lolitrem B and ergovaline were altered in response to this abiotic stress in a manner that was specific for each symbiotic association.
[Show abstract][Hide abstract] ABSTRACT: Recombinant ATP sulfurylase (AcATPS1) and adenosine-5'-phosphosulfate reductase (AcAPR1) from Allium cepa have been used to determine if these enzymes form protein-protein complexes in vitro. Using a solid phase binding assay, AcAPR1 was shown to interact with AcATPS1. The AcAPR1 enzyme was also expressed in E. coli as the N-terminal reductase domain (AcAPR1-N) and the C-terminal glutaredoxin domain (AcAPR1-C), but neither of these truncated proteins interacted with AcATPS1. The solid-phase interactions were confirmed by immune-precipitation, where anti-AcATPS1 IgG precipitated the full-length AcAPR1 protein, but not AcAPR1-N and AcAPR1-C. Finally, using the ligand binding assay, full-length AcATPS1 has been shown to bind to membrane-localised full-length AcAPR1. The significance of an interaction between chloroplastidic ATPS and APR in A. cepa is evaluated with respect to the control of the reductive assimilation of sulfate.
[Show abstract][Hide abstract] ABSTRACT: Laser photoacoustic spectroscopy continuously quantified the ethylene (C2H4) produced by strawberry flowers and fruits developing in planta. C2H4 was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower−1 h−1) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C2H4 production was maintained at a ‘low level’ (10–60 pl fruit−1 h−1) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit−1 h−1). After expansion, C2H4 production declined to a low level until fruit attained the red-ripe stage for at least 24 h. After this time, C2H4 levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit−1 h−1. Twenty-four hours after the re-initiation of C2H4 production by red fruit, CO2 levels increased approximately three-fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C2H4 production is regulated by negative feedback until fruits had expanded. The C2H4 produced by red-ripe fruit was regulated by positive feedback. Anti-1-amino-cyclopropane-1-carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (Mr) within the fruit achenes of expanding and red-ripe fruit. Analysis of dissected fruit showed that seed C2H4 accounts for 50% the C2H4 that is detectable from ripe fruit.
Physiologia Plantarum 05/2006; 127(2):247 - 259. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four 5' flanking sequences, comprising the 5'-UTR and upstream promoter region, have been isolated and cloned from the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family of white clover (Trifolium repens L.), and designated TR-ACO1p, TR-ACO2p, TR-ACO3p and TR-ACO4p. Southern analysis confirmed that these sequences correspond to four distinct genes. The four corresponding genomic sequences have also been isolated and each shown to be comprised of four exons interspersed by three introns. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into white clover. Here, the TR-ACO1 5' flanking sequence directs highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the TR-ACO2 5' flanking sequence directs expression in both younger, mature green and in ontologically ageing tissue. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. The TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, but all four 5' flanking sequences directed expression in the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule, with the primacy of each promoter determined by the ontological age of the tissues. These data suggest that in vegetative tissue development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.
[Show abstract][Hide abstract] ABSTRACT: Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.
[Show abstract][Hide abstract] ABSTRACT: We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.