Susanna Leung

Complex formation between recombinant ATP sulfurylase and APS reductase of Allium cepa (L.)

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.

Changes in 1-aminocyclopropane-1-carboxlate (ACC) oxidase expression and enzyme activity in response to excess manganese in white clover (Trifolium repens L.).

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.

Ethylene and the responses of plants to phosphate deficiency

This review considers the evidence that ethylene biosynthesis is up-regulated by locally-generated signals in response to a change in external P supply, where the hormone then mediates, with auxin, changes in root system architecture. Subsequent changes in endogenous P evoke systemic responses whereby ethylene again is important in inducing some of the key signature changes observed in P-deprived tissues (eg. phosphate transporter and acid phosphatase up-regulation).

Transcription of Biotic Stress Associated Genes in White Clover (Trifolium repens L.) Differs in Response to Cyst and Root-Knot Nematode Infection

The transcription of four members of the Kunitz proteinase inhibitor (KPI) gene family of white clover (Trifolium repens L.), designated as Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5, was investigated at both local infection (roots) and systemic (leaf tissue) sites in white clover in response to infection with the clover root knot nematode (CRKN) Meloidogyne trifoliophila and the clover cyst nematode (CCN) Heterodera trifolii. Invasion by the CRKN resulted in a significant decrease in transcript abundance of Tr-KPI4 locally at both 4 days post-infection (dpi) and at 8 dpi, and an increase in transcription of Tr-KPI1 systemically at 8 dpi. In contrast, an increase in transcript abundance of all four Tr-KPI genes locally at 4 and 8 dpi, and an increase of Tr-KPI1, Tr-KPI2, and Tr-KPI5 at 8 dpi systemically was observed in response to infection with the CCN. Challenge of a resistant (R) genotype and a susceptible (S) genotype of white clover with the CCN revealed a significant increase in transcript abundance of all four Tr-KPI genes locally in the R genotype, while an increase in abundance of only Tr-KPI1, Tr-KPI2, and Tr-KPI5 was observed in the S genotype, and only at 4 dpi. The transcript abundance of a member of the1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE gene family from white clover (Tr-ACS1) was significantly down-regulated locally in response to CRKN infection at 4 and 8 dpi and at 4 dpi, systemically, while abundance increased locally and systemically at 8 dpi in response to CCN challenge. Conversely, the abundance of the jasmonic acid (JA) signalling gene, CORONATINE-INSENSITIVE PROTEIN 1 from white clover (Tr-COI1) increased significantly at 8 dpi locally in response to CRKN infection, but decreased at 8 dpi in response to CCN infection. The significance of this differential regulation of transcription is discussed with respect to differences in infection strategy of the two nematode species.

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L.) Plants

The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs) are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover (Trifolium repens L.) Kunitz Proteinase Inhibitor (Tr-KPI) gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1, Tr-KPI2, and Tr-KPI5, was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1, a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs, particularly Tr-KPI5, have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.)

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.