Luit J. De Kok

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H2S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H2S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H2S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.

Atmospheric H2S as sulphur source for Brassica oleracea: consequences for the activity of the enzymes of the assimilatory sulphate reduction pathway

Short-term exposure of Brassica oleracea L. (curly kale) to atmospheric H2S levels (0.2–0.8 μL·L–1), which are sufficient to meet the plants sulphur requirement, resulted in a decrease in the activity of adenosine 5’-phosphosulphate reductase (APR) in the shoot. The reduction in APR activity was maximally 80 % and was already substantial after 1 d exposure to 0.2 μL·L–1 H2S. The activity of APR in the roots remained unaffected upon exposure to all levels of H2S. The activities of ATP-sulphurylase (ATPS), serine acetyltransferase (SAT) and O-acetylserine(thiol)lyase (OAS-TL), in both shoot and roots were not affected upon exposure to H2S levels ranging from 0.2–0.8 μL·L–1. There was a rapid increase in the shoot thiol content, including cysteine, upon H2S exposure and a maximal 3-fold increase in thiol content occurred after 5 h exposure. In the roots, the thiol content was only slightly increased after 2 d H2S exposure. The relationship between the pattern of thiol accumulation and changes in sulphate assimilation upon H2S exposure is discussed.

Determination of Cysteine and its Accumulation in Spinach Leaf Tissue upon Exposure to Excess Sulfur

Summary The cysteine level in plant leaf tissue was determined by measuring the difference in the sulfhydryl content of a deproteinized 30,000 g supernatant extract before and after reaction of the cysteine sulfhydryl with methylglyoxal. In leaves of red clover, sugar beet and spinach, cysteine accounted for 5, 23 and 19 % of the water-soluble non-protein sulfhydryl compounds, respectively. A 24 h exposure of spinach leaf tissue to excess sulfur in the form of atmospheric 0.25 μll −1 HZS or 0.33 μll −1 SO 2 or to 50 mM Na 2 SO 4 (liquid phase) resulted in an increase of the non-protein sulfhydryl levels in light and in darkness. The cysteine level increased relatively more than the total sulfhydryl content and it could account for more than 30% of the total sulfhydryl. Although glutathione was the major water-soluble non-protein sulfhydryl compound in spinach leaf tissue, the high levels of cysteine after exposure to excess sulfur indicate a rather poor metabolic control of intracellular levels of this compound.

Expression and activity of sulfate transporters and APS reductase in curly kale in response to sulfate deprivation and re-supply.

Both activity and expression of sulfate transporters and APS reductase in plants are modulated by the sulfur status of the plant. To examine the regulatory mechanisms in curly kale (Brassica oleracea L.), the sulfate supply was manipulated by the transfer of seedlings to sulfate-deprived conditions, which resulted in an up to 3-fold increase in the sulfate uptake capacity by the root, accompanied by an induction of transcript abundances of the Group 1 and 4 sulfate transporters in root and shoot. Upon sulfate re-supply, there was no correlation between the activity and expression of the sulfate transporters. Despite the decrease in the abundance of the sulfate transporter transcripts, especially at the onset of the sulfate re-supply, the sulfate uptake capacity was affected very little for up to 96h. There was no relationship between changes in the sulfate or thiol content and activity and expression of the sulfate transporters. Thus, their significance as regulatory signal compounds remains unresolved. The activity and expression of APS reductase, which was enhanced strongly only in the shoots of sulfate-deprived plants, and rapidly decreased again upon sulfate re-supply, corresponded with changes in thiol content, consistent with this pool having a role as a regulatory signal.

Copper exposure interferes with the regulation of the uptake, distribution and metabolism of sulfate in Chinese cabbage.

Exposure of Chinese cabbage (Brassica pekinensis) to enhanced Cu(2+) concentrations (1-10 microM) resulted in leaf chlorosis, a loss of photosynthetic capacity and lower biomass production at > or = 5 microM. The decrease in pigment content was likely not the consequence of degradation, but due to hindered chloroplast development upon Cu exposure. The Cu content of the root increased with the Cu(2+) concentration (up to 40-fold), though only a minor proportion (4%) was transferred to the shoot. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+). The nitrogen content of the root was affected little at lower Cu(2+) levels, whereas that in the shoot was decreased at > or = 5 microM Cu(2+). Cu affected the uptake, distribution and metabolism of sulfate in Chinese cabbage. The total sulfur content of the shoot was increased at > or = 2 microM Cu(2+), which could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in water-soluble non-protein thiol content in the root and, to a lesser extent, in the shoot at > or = 1 microM, which could only partially be ascribed to a Cu-induced enhancement of the phytochelatin content. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+), coinciding with a decrease in biomass production. However, the activity of the sulfate transporters in the root was slightly enhanced at 2 and 5 microM Cu(2+), accompanied by enhanced expression of the Group 1 high affinity transporter Sultr1;2, and the Group 4 transporters Sultr4;1 and Sultr4;2. In the shoot, there was an induction of expression of Sultr4;2 at 5 and 10 microM Cu(2+). The expression of APS reductase was affected little in the root and shoot up to 10 microM Cu(2+). The upregulation of the sulfate transporters may be due not only to greater sulfur demand at higher Cu levels, but also the consequence of interference by Cu with the signal transduction pathway regulating the expression and activity of the sulfate transporters.

Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana

In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by β-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.

Atmospheric H2S as sulfur source for Brassica oleracea: kinetics of H2S uptake and activity of O-acetylserine (thiol)lyase as affected by sulfur nutrition

The uptake of hydrogen sulfide (H(2)S) by shoots of curly kale (Brassica oleracea) showed saturation kinetics with respect to the atmospheric concentration. The kinetics are largely determined by the rate of metabolism of the absorbed H(2)S into cysteine, catalyzed by O-acetylserine (thiol)lyase, and can be described by the Michaelis-Menten equation. When B. oleracea was grown under sulfate (SO(4)(2-))-deprived conditions, plants developed sulfur (S) deficiency symptoms and H(2)S uptake kinetics were substantially altered. Shoots of SO(4)(2-)-deprived plants had a lower affinity to H(2)S uptake, whereas the maximal H(2)S uptake rate was higher. When SO(4)(2-)-deprived plants were simultaneously exposed to 0.2 &mgr;l l(-1) H(2)S all S deficiency symptoms disappeared and H(2)S uptake kinetics returned rapidly to values observed for S-sufficient shoots. The activity of the H(2)S-fixating enzyme O-acetylserine (thiol)lyase was hardly affected upon either prolonged H(2)S exposure or SO(4)(2-) deprivation. Evidently, the activity of O-acetylserine (thiol)lyase was not the rate-limiting step in the H(2)S uptake by shoots. The significance of the in situ availability and rate of synthesis of the substrate O-acetylserine for O-acetylserine (thiol)lyase as determining factor in the uptake kinetics of H(2)S needs further evaluation.

Effect of H2S Fumigation on Water-Soluble Sulfhydryl Compounds in Shoots of Crop Plants

Summary Long-term exposure of Spinacea oleracea and Beta vulgaris to 30, 100 and 300 ppb H 2 S resulted in reduced plant growth at 300 ppb and in an increased water-soluble sulfhydryl-compound content of shoots, even at an exposure to 30 ppb H 2 S. The increase in water-soluble sulfhydryl compounds was noticed during the entire 44 day fumigation period, in spite of ageing of the plants which caused a decrease of the actual sulfhydryl content of the control plants. The increase of sulfhydryl content in spinach was due to an accumulation of glutathione. Already after three days of exposure to 250 ppb H 2 S, the glutathione level in the shoot was increased more than four-fold. The significance of glutathione accumulation during H 2 S exposure for plant metabolism and growth is discussed.

The Effect of H2S Fumigation on Various Spinach (Spinacia oleracea L.) Cultivars Relation between growth inhibition and accumulation of sulphur compounds in the plant

Summary Several cultivars of spinach ( Spinacia oleracea L.) were continuously fumigated with 0.3 8 mg m −3 H 2 S. For the two cultivars tested, Subito and Dynamo, the rate of germination was not affected by H 2 S fumigation, but the total percentage of germinated seeds was slightly increased. Shoot growth of all ten cultivars tested, with the exception of cv. Monosa, was inhibited by 50 % on the average by H 2 S. Monosa was significantly less reduced than the other cultivars tested at a day and night temperature of 16/12 °C and more reduced at 18/18 °C. H 2 S reduced the growth in length of the cotyledon. This reduction was positively correlated with the reduction in growth of the total shoot. Dry matter content was slightly increased in the plants exposed to H 2 S. Water-soluble non-protein sulphydryl and sulphate content increased to various extents in both cotyledons and leaves. There was no direct relation between accumulation of sulphur compounds and reduction of growth.

Elevated Levels of Hydrogen Sulfide in the Plant Environment: Nutrient or Toxin

Hydrogen sulfide (H2S) is a malodorous gas with a typical “rotten egg” odor that can be smelt at levels of 0.02 μl l-1 and higher (for chemical and physical properties of H2S, see the review of Beauchamp et al. 1984). Normally H2S is only present in trace concentrations in the plant environment, but under specific conditions plants may have to cope with elevated levels of H2S in either the pedosphere or atmosphere in both natural vegetation and agriculture. Even though sulfide is a normal intermediate in plant metabolism, the impact of H2S on plants is paradoxical. On the one hand, it may be utilized as a sulfur nutrient, and on the other hand, above a certain threshold level it may negatively affect plant growth and functioning. In this chapter, our present knowledge on the impact of elevated levels of H2S on plants both as nutrient and toxin is reviewed.