Bok-Rye Lee

Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.

Diurnal and light regulation of sulphur assimilation and glucosinolate biosynthesis in Arabidopsis

Glucosinolates are a major class of sulphur-containing secondary metabolites involved in plant defence against pathogens. Recently many regulatory links between glucosinolate biosynthesis and sulphate assimilation were established. Since sulphate assimilation undergoes diurnal rhythm and is light regulated, this study analysed whether the same is true for glucosinolate biosynthesis. The levels of glucosinolates and glutathione were found to be higher during the day than during the night. This agreed with variation in sulphate uptake as well as activity of the key enzyme of the sulphate assimilation pathway, adenosine 5’-phosphosulphate reductase. Correspondingly, the flux through sulphate assimilation was higher during the day than during the night, with the maximum flux through primary assimilation preceding maximal incorporation into glucosinolates. Prolonged darkness resulted in a strong reduction in glucosinolate content. Re-illumination of such dark-adapted plants induced accumulation of mRNA for many genes of glucosinolate biosynthesis, leading to increased glucosinolate biosynthesis. The light regulation of the glucosinolate synthesis genes as well as many genes of primary sulphate assimilation was controlled at least partly by the LONG HYPOCOTYL5 (HY5) transcription regulator. Thus, glucosinolate biosynthesis is highly co-regulated with sulphate assimilation.

Water‐deficit accumulates sugars by starch degradation—not by de novo synthesis—in white clover leaves (Trifolium repens)

Labeling 13CO2 in steady-state condition was used to estimate quantitative mobilization of recently fixed carbon or stored sugar during water-deficit in white clover (Trifolium repens L.). Water-deficient gradually decreased leaf-water parameters and total amount of recently fixed carbon. Amount of 13C incorporated into glucose, sucrose and soluble sugars fraction rapidly decreased after 3 days of water-deficit treatment. In contrast, the previously stored soluble sugars significantly increased after 5 days of water-deficit with a coincidence of significant decrease in starch concentration. A highly significant (P < or = 0.001) relationship between the decrease in leaf-water potential caused by water-deficit and the increase in ratio of soluble sugar/starch concentration was observed in water deficit-stressed plants. The data indicate that soluble carbohydrate accumulated by water-deficit treatment is mainly because of the hydrolysis of previously stored starch rather than to de novo synthesis.

Control of sulfur partitioning between primary and secondary metabolism in Arabidopsis.

Sulfur is an essential nutrient for all organisms. Plants are able to take up inorganic sulfate and assimilate it into a range of bio-organic molecules either after reduction to sulfide or activation to 3′-phosphoadenosine 5′-phosphosulfate. While the regulation of the reductive part of sulfate assimilation and the synthesis of cysteine has been studied extensively in the past three decades, much less attention has been paid to the control of synthesis of sulfated compounds. Only recently the genes and enzymes activating sulfate and transferring it onto suitable acceptors have been investigated in detail with emphasis on understanding the diversity of the sulfotransferase gene family and the control of partitioning of sulfur between the two branches of sulfate assimilation. Here, the recent progress in our understanding of these processes will be summarized.

Effects of fou8/fry1 Mutation on Sulfur Metabolism: Is Decreased Internal Sulfate the Trigger of Sulfate Starvation Response?

The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5′phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3′-phosphoadenosine 5′-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism.

De novo protein synthesis in relation to ammonia and proline accumulation in water stressed white clover

The kinetics of protein incorporation from newly-absorbed nitrogen (N, de novo protein synthesis) was estimated by 15N tracing in 18-week-old white clover plants (Trifolium repens L. cv. Regal) during 7 d of water-deficit treatment. The physiological relationship between kinetics and accumulation of proline and ammonia in response to the change in leaf-water parameters was also assessed. All leaf-water parameters measured decreased gradually under water deficit. Leaf and root dry mass was not significantly affected during the first 3 d when decreases in leaf-water parameters were substantial. However, metabolic parameters such as total N, proline and ammonia were significantly affected within 1 d of commencement of water-deficit treatment. Water-deficit treatment significantly increased the proline and NH3-NH4+ concentrations in both leaves and roots. There was a marked reduction in the amount of N incorporated into the protein fraction from the newly absorbed N (NANP) in water-deficit stressed plants, particularly in leaf tissue. This reduction in NANP was strongly associated with an increased concentration of NH3-NH4+ in roots (P≤0.05) and proline (P≤0.01) in leaves and roots. These results suggest that proline accumulation may be a sensitive biochemical indicator of plant water status and of the dynamics of de novo protein synthesis in response to stress severity.

Natural Variation in the ATPS1 Isoform of ATP Sulfurylase Contributes to the Control of Sulfate Levels in Arabidopsis

Variation in mRNA levels for a single isoform of ATP sulfurylase in Arabidopsis affects the total enzyme activity and sulfate assimilation and causes a variation in foliar sulfate levels. Sulfur is an essential macronutrient for all living organisms. Plants take up inorganic sulfate from the soil, reduce it, and assimilate it into bioorganic compounds, but part of this sulfate is stored in the vacuoles. In our first attempt to identify genes involved in the control of sulfate content in the leaves, we reported that a quantitative trait locus (QTL) for sulfate content in Arabidopsis (Arabidopsis thaliana) was underlain by the APR2 isoform of the key enzyme of sulfate assimilation, adenosine 5′-phosphosulfate reductase. To increase the knowledge of the control of this trait, we cloned a second QTL from the same analysis. Surprisingly, the gene underlying this QTL encodes the ATPS1 isoform of the enzyme ATP sulfurylase, which precedes adenosine 5′-phosphosulfate reductase in the sulfate assimilation pathway. Plants with the Bay allele of ATPS1 accumulate lower steady-state levels of ATPS1 transcript than those with the Sha allele, which leads to lower enzyme activity and, ultimately, the accumulation of sulfate. Our results show that the transcript variation is controlled in cis. Examination of ATPS1 sequences of Bay-0 and Shahdara identified two deletions in the first intron and immediately downstream the gene in Bay-0 shared with multiple other Arabidopsis accessions. The average ATPS1 transcript levels are lower in these accessions than in those without the deletions, while sulfate levels are significantly higher. Thus, sulfate content in Arabidopsis is controlled by two genes encoding subsequent enzymes in the sulfate assimilation pathway but using different mechanisms, variation in amino acid sequence and variation in expression levels.

Water deficit-induced oxidative stress and the activation of antioxidant enzymes in white clover leaves

The objective of this study was to determine the development of the antioxidant enzymes induced by drought stress in white clover (Trifolium repens L.) leaves. Water stress was imposed during 28 d by decreasing the daily irrigation. Leaf water potential (Φw) gradually decreased from −0.46 to −2.33 MPa. For the first 7 d, dry mass (DM), H2O2 and lipid peroxidation were not significantly affected by water deficit. From 14 d of treatment, water stress decreased dry mass and increased content of reactive oxygen species (O2·− and H2O2) and oxidative stress (malondialdehyde content). The ascorbate peroxidase (APOD) was activated most rapidly, already during the first week of water stress, but then its activity slowly decreased. Activation of superoxide dismutase (SOD) and catalase (CAT) by water deficit continued during the 14 d (Φw ≥ −1.65 MPa) and then their activities remain on the similar level. The activity of guaiacol-peroxidase (GPOD) increased mostly under progressive water stress and was correlated with increase in lipid peroxidation and growth restriction.

Increased proline loading to phloem and its effects on nitrogen uptake and assimilation in water-stressed white clover (Trifolium repens).

The aim of this study was to investigate the physiological significance of increased proline loading to phloem caused by water-deficit stress in relation to nitrogen (N) uptake and assimilation. N uptake and N assimilation were quantified by 15N tracing in well-watered (control) and water deficit-stressed white clover (Trifolium repens). De novo proline synthesis and proline loading to the phloem were also compared between treatments. The relationships among proline concentrations in phloem exudates, N uptake, and assimilation of newly absorbed N were assessed. The newly synthesized proline in the phloem exudates increased rapidly after 3 d of water deficit. The water-deficit treatment significantly reduced the maximum nitrate reductase activity (NRA), and also attenuated de novo synthesis of amino acids and proteins in the roots. The increase in proline concentrations in phloem exudates was closely related to reductions in NRA in the roots, N uptake, and the assimilation of newly absorbed N. The accumulation of proline induced in roots by exogenous proline and NH4Cl treatments was closely associated with the decrease in NRA. These results indicate that increased proline transport to roots via phloem caused by water deficit has a significant influence on the down-regulation of N uptake and the assimilation of newly absorbed N.

Kinetics of drought-induced pathogenesis-related proteins and its physiological significance in white clover leaves.

To investigate the responses of pathogenesis-related (PR) proteins to the intensity of drought stress and their physiological significance in white clover (Trifolium repens L.), the change of enzyme activity and its relationship with some physiological parameters were assessed for 28 days under well-watered (control) and water-deficit conditions. Water-deficit treatment gradually decreased leaf water potential (Psi(w)) to -2.33 MPa at day 28. Dry matter significantly decreased from 21 days of water-deficit treatment, while proline and ammonia concentration increased within 7 days. The increase in PR-protein activity was closely related with the decrease in Psi(w). The beta-1,3-glucanase (EC 3.2.1.39) activity in water-deficit leaves rapidly increased for the first 14 days (Psi(w) >or= -1.67) and then slightly decreased, while the chitinase (EC 3.2.1.14) and cellulase (EC 3.2.1.4) activity continued to increase throughout the experimental period. The enhanced activation of beta-1,3-glucanase, chitinase and cellulase for the period of days 0-14 was significantly (P <or= 0.01) related to the increase of proline and ammonia concentrations. The results indicate that the enhanced activity of beta-1,3-glucanase, cellulase and chitinase for the early period might be an act of transient tolerance to drought stress, but the activation of these enzymes during terminal stress might be a drought-stress-induced injurious symptom.