Figure 1.
Scheme of involvement of FOU8 in sulfur metabolism.
Figure 2.
fou8 is affected in glucosinolate synthesis.
Col-0, fou8, apk1 apk2, and fou8 apk1 apk2 plants were grown for 5 weeks in controlled environment room. The total content of A glucosinolates and B desulfo-glucosinolates was measured in leaves. C Total RNA was isolated from leaves and the transcript levels of six genes involved in glucosinolate synthesis was determined by quantitative RT-PCR. The qRT-PCR reactions were performed in triplicate for each biological sample. The values in Col-0 were set to 1 for all genes. Results are presented as means ± SE from six pools of three individual plants grown in two independent experiments. Different letters mark values significantly different at P<0.05; asterisks mark values significantly different from Col-0 at P<0.05.
Table 1.
Levels of individual glucosinolates in Col-0 and fou8.
Figure 3.
Glucosinolate synthesis in fou8 mutant with and without cytosolic APS kinase.
Col-0, fou8, apk3, and fou8 apk3 plants were grown for 5 weeks in controlled environment room. The total content of A glucosinolates and B desulfo-glucosinolates was measured in leaves. C Total RNA was isolated from leaves and the transcript levels of six genes involved in glucosinolate synthesis was determined by quantitative RT-PCR. The qRT-PCR reactions were performed in triplicate for each biological sample. The values in Col-0 were set to 1 for all genes. Results are presented as means ± SE from six pools of three individual plants grown in two independent experiments. Different letters mark values significantly different at P<0.05; asterisks mark values significantly different from Col-0 at P<0.05.
Table 2.
Relative expression of sulfate assimilation genes in fou8.
Figure 4.
Accumulation of sulfur-containing compounds in fou8.
Col-0 and fou8 plants were grown for 5 weeks in controlled environment room. Leaves were harvested and A APR activity was determined. Levels of B cysteine C glutathione, and D sulfate were measured by HPLC. Results are presented as means ± SE from six individual plants, grown in two independent experiments. Asterisks mark values significantly different from Col-0 at P<0.05.
Table 3.
Mineral content of Col-0 and fou8.
Figure 5.
Comparison of mRNA levels of key genes of sulfur metabolism.
Col-0, fou8, cad2, rax1, and sultr1;2 plants were grown for 2.5 weeks on MS-agarose plates. Relative mRNA levels of ATPS4 and APR1 in A leaves and B roots and of LS2 (At5g48850) and LS5 (At5g26220) in C leaves and D roots and genes of glucosinolate synthesis in E leaves were determined. The qRT-PCR reactions were performed in triplicate, the values in Col-0 were set to 1 for all genes. Results are presented as means ± SE from six biological replicates from plants grown in two independent experiments. Different letters mark values significantly different at P<0.05 or in E asterisks show values significantly (P<0.05) different from Col-0.
Table 4.
Contents of sulfur-containing metabolites in genotypes affected in sulfur metabolism.
Figure 6.
Variation in fatty acid oxygenation rate in fou8 related genotypes.
The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Leaf juice was incubated for 2 min with 1-[14C]18∶3. Products were separated by thin layer chromatography and the radioactivity quantified. The rate of oxygenation is expressed as % of radioactivity in 18∶3-α-ketol. Results are presented as means ± SE from six individual plants grown in two independent experiments, different letters mark values significantly different at P<0.05.
Figure 7.
Accumulation of sulfur-containing compounds in fou8 related genotypes.
The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Leaves were harvested and the levels of sulfur-containing compounds were determined by HPLC. A sulfate, B glutathione, C glucosinolates, and D desulfo-glucosinolates. Results are presented as means ± SE from six individual plants grown in two independent experiments with three replicates each. Different letters mark values significantly different at P<0.05; ND = not detectable.
Figure 8.
Expression analysis of Arabidopsis lines.
The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Total RNA was isolated from leaves and the transcript levels of genes involved in sulfur metabolism, glucosinolate synthesis, and jasmonate synthesis were determined by quantitative RT-PCR. The qRT-PCR reactions were performed in triplicate for each of the six independent biological samples from plants grown in two independent experiments. Results are presented as a heat map of relative mRNA levels compared to Col-0. For comparison, sulfate levels are presented in the same way on the far right.
Figure 9.
Accumulation of PAP and PAPS in fou8 related genotypes.
The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Leaves were harvested and the levels of A PAP and B PAPS were determined by HPLC. Results are presented as means ± SD from four individual plants, grown in two independent experiments. Asterisks represents levels under detection limit, different letters mark values significantly different at P<0.05.
Figure 10.
Sulfate uptake and flux through sulfate assimilation in fou8 and related mutants.
WT Col-0 and mutants fou8, fou2, and aos were grown for 3 weeks on MS-phytagel vertical plates in controlled environment room. The seedlings were incubated for four hours with their roots submerged in nutrient solution adjusted to sulfate concentration of 0.2 mM and supplemented with 6.7 μCi [35S]sulfate. Shoot and root material was harvested separately, and the flux was determined as incorporation of 35S from [35S] sulfate to thiols and proteins. A sulfate uptake, B Percentage of 35S transported to leaves from the [35S]sulfate taken up, C relative flux through the sulfate assimilation in the leaves calculated as % of incorporation in thiols and proteins from total [35S]sulfate taken up. Results are presented as means ± SE from six independent pools of 8 seedlings grown in two independent experiments. Values marked with an asterisk show significant (P≤0.05) difference from Col-0.