Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves

Abstract

Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants.

Keywords: carbon metabolism; dark-induced senescence; lipid degradation; nitrogen; sulfite oxidase; sulfur.

Figure 1

Turnover of sulfur containing metabolites in wild-type (Wt) and sulfite oxidase (SO) RNA interference mutants (Ri) plants grown under normal growth conditions (0 day) and after being exposed to an extended dark stress for eleven days (11 day). The Log2 of sulfur containing metabolites are given by shades of red or blue colors according to scale bar. (a) Relative difference of sulfur-containing metabolites in SO Ri mutants calculated as ratio to WT 0 day; (b) The relative difference of S-containing metabolites was calculated as the ratio of metabolite content in plants after the dark stress (11 day) to the content detected in the unstressed plants (0 day). The other S-compounds metabolites were calculated as the difference between total content of sulfur to the known S-containing metabolites. The organic S was calculated as the difference between total S to the inorganic S (oxidized + reduced). The level of significance was estimated with Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05. n = 3–10) and is presented in Supplementary Table S2. The data for SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 2

Energy-source metabolites in wild-type (Wt) and SO RNA interference mutants (Ri) plants grown under normal growth conditions (0 day) and after being exposed to eleven days of dark stress (11 day). Top leaves of WT and Ri tomato plants were used to determine total carbon levels (a). Relative carbohydrate, sugars sucrose (b); fructose (c); sugar alcohols; sorbitol (d) and myoinositol (e) contents were also determined. The bars represent the average values ± SE (n = 3–8). Values denoted with different letters are significantly different according to the Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05). Different lower case letters indicate differences between the SO mutants and wild-type plants under the same treatment. Different upper case letters indicate significant differences within the plant genotypes in response to treatment. The data for the SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 3

The levels of pyruvate, the product of glycolysis, and tricarboxylic acid cycle (TCA) cycle intermediates in wild-type (Wt) and SO RNA interference mutants (Ri) plants grown under normal growth conditions (0, day) and after being exposed to eleven days of extended dark stress (11, day). Top leaves of Wt and Ri tomato plants were used to determine pyruvate (a); citrate (b); a-ketoglutarate (c); succinate (d); fumarate (e) and malate (f) content. The bars are the average values ± SE (n = 3–8). The values denoted with different letters are significantly different according to the Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05). Different lower case letters indicate differences between the SO mutants and wild-type plants within the same treatment. Different upper case letters indicate significant difference within the plant genotypes in response to treatment. The data for SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 4

Lipid content in tomato wild-type (Wt) and SO RNA interference mutant (Ri) plants grown under normal growth conditions (0, day) and after being exposed to eleven days extended dark stress (11, day). Total polar lipids (a), total galactolipids (b) and individual galactolipids, (c,d) as well as the ratio between the galactolipids (e), total phospholipids (f) and individual phospholipids (g–i) were quantified by Trace GC Ultra as their methyl esters of the constituent fatty acids after two-dimensional thin-layer chromatography. First dimension was chlorophorm:Methanol:H₂O (65:25:4, v/v/v) followed byChlorophorm:Methanol:NH₄OH: Isopropylamine (65:35:5:0.5, v/v/v/v) and visualization under UV. Abbreviations are: digalactosyl-diacylglycerol (DGDG); monogalactosyl-diacylglycerol (MGDG); phosphatidylglycerol (PG); phosphatidylcholine (PC); phosphatidylethanolamine (PE). The bars are the average values ± SE (n = 3). The values denoted with different letters are significantly different according to the Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05). Different lower case letters indicate differences between SO mutant and wild-type plants within the same treatment. Different upper case letters indicate significant difference within the plant genotypes in response to treatment. The data for SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 5

Heat map of free and protein bound amino acids in wild-type (WT) and SO RNA interference mutant (Ri) plants grown under normal growth conditions (0, day) and after being exposed to eleven days of extended dark stress (11, day). Log2 ratios of bound (a) and free (b) amino acids (AA) fold-changes from WT 0 day are given by shades of red or blue colors according to the scale bar; (c) Log2 ratios of residual to initial total AA were calculated as the Log2 ratio of total AA at 11 day to 0 day and are given by shades of red or blue colors according to scale bar. Asp—aspartate; Lys—lysine; Met—methionine; Thr—threonine; Glu—glutamate; Pro—proline; Cys—cysteine; Gly—glycine; Ser—serine; Ala—alanine; Leu—leucine; Val—valine; Phe—phenylalanine; Tyr—tyrosine; His—histidine. Data represent the mean value of three (WT) and six (Ri) biological replicates for each tissue and time point and statistical analysis performed using Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05), presented in Supplementary Table S3. The data for SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 6

The levels of the ureides (a) allantoin and (b) allanoate and the amides (c) glutamine (Gln) and (d) asparagine (Asn) in wild-type (Wt) and SO RNA interference mutants (Ri) plants. Plants were grown under normal growth conditions (0 day) or after being exposed to eleven days of extended dark stress (11 day). The bars are the average values ± SE (n = 3–10). The values denoted with different letters are significantly different according to the Turkey-Kramer HSD test (JMP 8.0 software, [37]; p < 0.05). Different lower case letters indicate differences between SO mutant and wild-type plants within the same treatment. Different upper case letters indicate a significant difference within the plant genotypes in response to treatment. The data for SO-compromised plants represent the mean for SO Ri 131 and SO Ri 421 mutants.

Figure 7

Schematic illustration describing the impact of sulfite oxidase (SO) impairment on the sulfur, carbon and nitrogen metabolism in tomato plants grown under normal growth conditions. Abbreviations are: adenosine-5′-phosphosulfate (APS), APS reductase (APR), sulfite reductase (SiR) O-acetyl-serine-thiol-lyase (OAS-TL), UDP-sulfoquinovose synthase (SQD1), sulfite oxidase (SO), sulfurtransferases (ST). The Cys Met box indicates the sum of total Cys and Met. The other AA indicates the total detected amino acids other than Cys and Met. Energy rich carbon indicates fatty acids, sucrose, fructose, sorbitol and myoinosytol. TCA indicates pyruvate and the following TCA cycle metabolites: citrate, α-ketoglutarate, succinate, fumarate and malate. Nitrogen indicates the total N as in Table 2. Organic S indicates total organic S as in Supplementary Table S2. ROS indicates the reactive oxygen species as in Supplementary Figure S3. Red and green colored boxes indicate an enhancement or a reduction in metabolite content, respectively, as compared to those in the unstressed top leaves of the wild-type plants. The non-colored metabolites indicate similar levels as compared to the unstressed top leaves of the wild-type plants. The red arrow indicates enhanced SiR activity as compared to wild-type. The grey arrow indicates decreased sulfite oxidation activity due to SO impairment.

Figure 8

Schematic illustration describing the impact of sulfite oxidase (SO) impairment on the sulfur, carbon and nitrogen metabolism in the top leaves of tomato plants grown for 11 days in dark. Abbreviations are: adenosine-5′-phosphosulfate (APS), APS reductase (APR), sulfite reductase (SiR) O-acetyl-serine-thiol-lyase (OAS-TL), UDP-sulfoquinovose synthase (SQD1), sulfite oxidase (SO), sulfurtransferases (ST). The Cys Met box indicates the sum of total Cys and Met. The other AA indicates the total detected amino acids other than Cys and Met. Energy rich carbon indicates fatty acids, sucrose, fructose, sorbitol and myoinosytol. TCA indicates pyruvate and the following TCA cycle metabolites: citrate, α-ketoglutarate, succinate, fumarate and malate. Nitrogen indicates the total N as in Table 2. Organic S indicates total organic S as in Supplementary Table S2. ROS indicates the reactive oxygen species as in Supplementary Figure S3. The blue box indicates that the sulfate level was not affected by the stress but was significantly lower in the mutant as compared with the stressed wild-type top leaves [14]. Red or green colors indicate the enhancement or reduction, respectively, in metabolites, as compared to the contents in the unstressed top leaves of the wild-type plants (light green indicates less reduction compared to the darker green). The red arrow from the Cys Met box indicates the direction of sulfite, the degradation product of these amino acids [14]. Red or grey arrows indicate the SO activity enhancement or decrease, respectively. The lack of colored metabolites or colored arrows indicates similar levels as compared to the unstressed top leaves of the wild-type plants.

Figure 8

Schematic illustration describing the impact of sulfite oxidase (SO) impairment on the sulfur, carbon and nitrogen metabolism in the top leaves of tomato plants grown for 11 days in dark. Abbreviations are: adenosine-5′-phosphosulfate (APS), APS reductase (APR), sulfite reductase (SiR) O-acetyl-serine-thiol-lyase (OAS-TL), UDP-sulfoquinovose synthase (SQD1), sulfite oxidase (SO), sulfurtransferases (ST). The Cys Met box indicates the sum of total Cys and Met. The other AA indicates the total detected amino acids other than Cys and Met. Energy rich carbon indicates fatty acids, sucrose, fructose, sorbitol and myoinosytol. TCA indicates pyruvate and the following TCA cycle metabolites: citrate, α-ketoglutarate, succinate, fumarate and malate. Nitrogen indicates the total N as in Table 2. Organic S indicates total organic S as in Supplementary Table S2. ROS indicates the reactive oxygen species as in Supplementary Figure S3. The blue box indicates that the sulfate level was not affected by the stress but was significantly lower in the mutant as compared with the stressed wild-type top leaves [14]. Red or green colors indicate the enhancement or reduction, respectively, in metabolites, as compared to the contents in the unstressed top leaves of the wild-type plants (light green indicates less reduction compared to the darker green). The red arrow from the Cys Met box indicates the direction of sulfite, the degradation product of these amino acids [14]. Red or grey arrows indicate the SO activity enhancement or decrease, respectively. The lack of colored metabolites or colored arrows indicates similar levels as compared to the unstressed top leaves of the wild-type plants.