Muhammad Shahbaz

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.

Copper toxicity in Chinese cabbage is not influenced by plant sulphur status, but affects sulphur metabolism-related gene expression and the suggested regulatory metabolites

The toxicity of high copper (Cu) concentrations in the root environment of Chinese cabbage (Brassica pekinensis) was little influenced by the sulphur nutritional status of the plant. However, Cu toxicity removed the correlation between sulphur metabolism-related gene expression and the suggested regulatory metabolites. At high tissue Cu levels, there was no relation between sulphur metabolite levels viz. total sulphur, sulphate and water-soluble non-protein thiols, and the expression and activity of sulphate transporters and expression of APS reductase under sulphate-sufficient or-deprived conditions, in the presence or absence of H2 S. This indicated that the regulatory signal transduction pathway of sulphate transporters was overruled or by-passed upon exposure to elevated Cu concentrations.

Interactions of sulfate with other nutrients as revealed by H2S fumigation of Chinese cabbage

Sulfur deficiency in plants has severe impacts on both growth and nutrient composition. Fumigation with sub-lethal concentrations of H2S facilitates the supply of reduced sulfur via the leaves while sulfate is depleted from the roots. This restores growth while sulfate levels in the plant tissue remain low. In the present study this system was used to reveal interactions of sulfur with other nutrients in the plant and to ascertain whether these changes are due to the absence or presence of sulfate or rather to changes in growth and organic sulfur. There was a complex reaction of the mineral composition to sulfur deficiency, however, the changes in content of many nutrients were prevented by H2S fumigation. Under sulfur deficiency these nutrients accumulated on a fresh weight basis but were diluted on a dry weight basis, presumably due to a higher dry matter content. The pattern differed, however, between leaves and roots which led to changes in shoot to root partitioning. Only the potassium, molybdenum and zinc contents were strongly linked to the sulfate supply. Potassium was the only nutrient amongst those measured which showed a positive correlation with sulfur content in shoots, highlighting a role as a counter cation for sulfate during xylem loading and vacuolar storage in leaves. This was supported by an accumulation of potassium in roots of the sulfur-deprived plants. Molybdenum and zinc increased substantially under sulfur deficiency, which was only partly prevented by H2S fumigation. While the causes of increased molybdenum under sulfur deficiency have been previously studied, the relation between sulfate and zinc uptake needs further clarification.

Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in hydroponics

Arable soils are frequently subjected to contamination with copper as the consequence of imbalanced fertilization with manure and organic fertilizers and/or extensive use of copper-containing fungicides. In the present study, the exposure of stone-head cabbage (Brassica oleracea var. capitata) to elevated Cu2+ levels resulted in leaf chlorosis and lesser biomass yield at ≥2 µ M. Root nitrate content was not statistically affected by Cu2+ levels, although it was substantially decreased at ≥5 µ M Cu2+ in the shoot. The decrease in nitrate contents can be related to lower nitrate uptake rates because of growth inhibition by Cu-toxicity. Shoot sulfate content increased strongly at ≥2 µ M Cu2+ indicating an increase in demand for sulfur under Cu stress. Furthermore, at ≥2 µM concentration, concentration of water-soluble non-protein thiol increased markedly in the roots and to a smaller level in the shoot. When exposed to elevated concentrations of Cu2+ the improved sulfate and water-soluble non-protein thiols need further studies for the evaluation of their direct relation with the synthesis of metal-chelating compounds (i.e., phytochelatins).

Regulation of Expression of Sulfate Transporters and APS Reductase in Leaf Tissue of Chinese Cabbage (Brassica pekinensis)

Leaf discs from sulfur-sufficient and sulfate-deprived Chinese cabbage plants were incubated at various levels of sulfate (ranging from 0 to 40 mM) for 24 h in the light. Sultr4;1 was the sole constitutively expressed sulfate transporter present in sulfur-sufficient leaf discs and its expression was enhanced upon incubation at ≤8 mM and decreased at higher sulfate concentrations. Leaf discs from sulfate-deprived plants were characterized by a high expression of Sultr1;2, Sultr4;1, Sultr4;2 and APS reductase. The high expression was down-regulated upon incubation of the leaf discs at >2 mM sulfate, whereas at 20–30 mM their expression was quite similar to that of sulfur-sufficient leaf discs. In both sulfur-sufficient and sulfur-deprived leaf discs there was an accumulation of sulfate upon incubation nearly linear with the sulfate concentration. The thiol content of leaf discs was slightly enhanced upon incubation at high sulfate concentrations. Evidently, the expression of the sulfate transporters and APS reductase were strictly controlled by the in situ sulfate concentration in the leaf tissue possibly via an interaction with the sulfate reduction in the chloroplasts. It is suggested that H2S might function as endogenous gaseous transmitter in cross-talk between the sulfate reduction pathway in the chloroplasts and the transcription of sulfate transporters/sulfate reducing enzymes in the nucleus.

Regulation of Cu delivery to chloroplast proteins

Plastocyanin is a copper (Cu)-requiring protein that functions in photosynthetic electron transport in the thylakoid lumen of plants. To allow plastocyanin maturation, Cu must first be transported into the chloroplast stroma by means of the PAA1/HMA6 transporter and then into the thylakoid lumen by the PAA2/HMA8 transporter. Recent evidence indicated that the chloroplast regulates Cu transport into the thylakoids via Clp protease-mediated turnover of PAA2/HMA8. Here we present further genetic evidence that this regulatory mechanism for the adjustment of intra-cellular Cu distribution depends on stromal Cu levels. A key transcription factor mediating Cu homeostasis in plants is SQUAMOSA promoter binding protein-like7 (SPL7). SPL7 transcriptionally regulates Cu homeostasis when the nutrient becomes limiting by up-regulating expression of Cu importers at the cell membrane, and down-regulating expression of seemingly non-essential cuproproteins. It was proposed that this latter mechanism favors Cu delivery to the chloroplast. We propose a 2-tiered system which functions to control plant leaf Cu homeostasis: SPL7 dependent transcriptional regulation of cuproproteins, and PAA2/HMA8 turnover by the Clp system, which is independent on SPL7.

Prioritization of copper for the use in photosynthetic electron transport in developing leaves of hybrid poplar

Plastocyanin (PC) is an essential and abundant copper (Cu) protein required for photosynthesis in higher plants. Severe copper deprivation has the potential to cause a defect in photosynthetic electron transport due to a lack in PC. The Cu-microRNAs, which are up-regulated under Cu deficiency, down-regulate the expression of target Cu proteins other than PC, cytochrome-c oxidase and the ethylene receptors. It has been proposed that this mechanism saves Cu for PC maturation. We aimed to test how hybrid poplar, a species that has capacity to rapidly expand its photosynthetically active tissue, responds to variations in Cu availability over time. Measurement of chlorophyll fluorescence after Cu depletion revealed a drastic effect on photosynthesis in hybrid poplar. The decrease in photosynthetic capacity was correlated with a reduction in PC protein levels. Compared to older leaves, PC decreased more strongly in developing leaves, which also lost more photosynthetic electron transport capacity. The effect of Cu depletion on older and more developed leaves was minor and these leaves maintained much of their photosynthetic capacity. Interestingly, upon resupply of Cu to the medium a very rapid recovery of Cu levels was seen in the younger leaves with a concomitant rise in the expression and activity of PC. In contrast, the expression of those Cu proteins, which are targets of microRNAs was under the same circumstances delayed. At the same time, Cu resupply had only minor effects on the older leaves. The data suggest a model where rapid recovery of photosynthetic capacity in younger leaves is made possible by a preferred allocation of Cu to PC in younger leaves, which is supported by Cu-microRNA expression.

Impact of Enhanced Copper Levels and Sulfate Deprivation on the Uptake and Metabolism of Sulfate in Chinese Cabbage (Brassica pekinensis)

Exposure of Chinese cabbage (Brassica pekinensis) to an enhanced Cu2+ level (4 μM) resulted in a reduced plant biomass production and an increased shoot to root ratio at both sulfate-sufficient and sulfate-deprived conditions. However, sulfate deprivation had a more rapid negative effect on plant biomass production than an enhanced Cu2+ level, The expression and activity of the sulfate transporters and the expression of APS reductase in Chinese cabbage were rapidly up-regulated (already after 1 or 2 days) upon exposure to 4 μM Cu2+, sulfate deprivation and their combination. Though the impact of sulfate deprivation on the expression and activity of the sulfate transporters was hardly further affected by Cu2+.