A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

Extensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologs of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal, suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery

Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism

A systems approach has previously been used to follow the response behaviour of Arabidopsis thaliana plants upon sulphur limitation. A response network was reconstructed from a time series of transcript and metabolite profiles, integrating complex metabolic and transcript data in order to investigate a potential causal relationship. The resulting scale-free network allowed potential transcriptional regulators of sulphur metabolism to be identified. Here, three sulphur-starvation responsive transcription factors, IAA13, IAA28, and ARF-2 (ARF1-Binding Protein), all of which are related to auxin signalling, were selected for further investigation. IAA28 overexpressing and knock-down lines showed no major morphological changes, whereas IAA13- and ARF1-BP-overexpressing plants grew more slowly than the wild type. Steady-state metabolite levels and expression of pathway-relevant genes were monitored under normal and sulphate-depleted conditions. For all lines, changes in transcript and metabolite levels were observed, yet none of these changes could exclusively be linked to sulphur stress. Instead, up- or down-regulation of the transcription factors caused metabolic changes which in turn affected sulphur metabolism. Auxin-relevant transcription factors are thus part of a complex response pattern to nutrient starvation that serve as coordinators of the metabolic shifts driving sulphur homeostasis rather then as direct effectors of the sulphate assimilation pathway. This study provides the first evidence ever presented that correlates auxin-related transcriptional regulators with primary plant metabolism

The C-Terminal Region of SLIM1 Transcription Factor Is Required for Sulfur Deficiency Response

Sulfur LIMitation1 (SLIM1) transcription factor coordinates gene expression in plants in response to sulfur deficiency (-S). SLIM1 belongs to the family of plant-specific EIL transcription factors with EIN3 and EIL1, which regulate the ethylene-responsive gene expression. The EIL domains consist of DNA binding and dimerization domains highly conserved among EIL family members, while the N- and C-terminal regions are structurally variable and postulated to have regulatory roles in this protein family, such that the EIN3 C-terminal region is essential for its ethylene-responsive activation. In this study, we focused on the roles of the SLIM1 C-terminal region. We examined the transactivation activity of the full-length and the truncated SLIM1 in yeast and Arabidopsis. The full-length SLIM1 and the truncated form of SLIM1 with a deletion of C-terminal 106 amino acids (dC105) transactivated the reporter gene expression in yeast when they were fused to the GAL4 DNA binding domain, whereas the deletion of additional 15 amino acids to remove the C-terminal 120 amino acids (dC120) eliminated such an activity, identifying the necessity of that 15-amino-acid segment for transactivation. In the Arabidopsis slim1-2 mutant, the transcript levels of SULTR1;2 sulfate transporter and the GFP expression derived from the SULTR1;2 promoter-GFP (PSULTR1;2-GFP) transgene construct were restored under -S by introducing the full-length SLIM1, but not with the C-terminal truncated forms dC105 and dC57. Furthermore, the transcript levels of S-responsive genes were restored concomitantly with an increase in glutathione accumulation in the complementing lines with the full-length SLIM1 but not with DC57. The C-terminal 57 amino acids of SLIM1 were also shown to be necessary for transactivation of a S-inducible gene, SHM7/MSA1, in a transient expression system using the SHM7/MSA1 promoter-GUS as a reporter. These findings suggest that the C-terminal region is essential for the SLIM1 activity

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a ‘sulphur-free’ solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance

Sulphur limitation and early sulphur deficiency responses in poplar: significance of gene expression, metabolites, and plant hormones

The influence of sulphur (S) depletion on the expression of genes related to S metabolism, and on metabolite and plant hormone contents was analysed in young and mature leaves, fine roots, xylem sap, and phloem exudates of poplar (Populus tremula×Populus alba) with special focus on early consequences. S depletion was applied by a gradual decrease of sulphate availability. The observed changes were correlated with sulphate contents. Based on the decrease in sulphate contents, two phases of S depletion could be distinguished that were denominated as ‘S limitation’ and ‘early S deficiency’. S limitation was characterized by improved sulphate uptake (enhanced root-specific sulphate transporter PtaSULTR1;2 expression) and reduction capacities (enhanced adenosine 5′-phosphosulphate (APS) reductase expression) and by enhanced remobilization of sulphate from the vacuole (enhanced putative vacuolar sulphate transporter PtaSULTR4;2 expression). During early S deficiency, whole plant distribution of S was impacted, as indicated by increasing expression of the phloem-localized sulphate transporter PtaSULTR1;1 and by decreasing glutathione contents in fine roots, young leaves, mature leaves, and phloem exudates. Furthermore, at ‘early S deficiency’, expression of microRNA395 (miR395), which targets transcripts of PtaATPS3/4 (ATP sulphurylase) for cleavage, increased. Changes in plant hormone contents were observed at ‘early S deficiency’ only. Thus, S depletion affects S and plant hormone metabolism of poplar during ‘S limitation’ and ‘early S deficiency’ in a time series of events. Despite these consequences, the impact of S depletion on growth of poplar plants appears to be less severe than in Brassicaceae such as Arabidopsis thaliana or Brassica sp

Establishment of an in planta magnesium monitoring system using CAX3 promoter-luciferase in Arabidopsis

The direct determination of elemental concentrations in plants is laborious. To overcome this, a novel monitoring system for magnesium (Mg) in plants was established. Mg deficiency-induced genes were identified by microarray analysis and transgenic lines that expressed luciferase (LUC) under the control of the Mg deficiency-inducible CAX3 promoter were established. The transgenic lines showed a clear response under low Mg conditions, and the degree of luminescence reflected the accumulation of endogenous CAX3 mRNA. The CAX3 expression pattern was also examined in a previously characterized low Mg-sensitive mutant, mrs2-7. In mrs2-7 mutant plants, CAX3 expression was more than three times higher than in the wild-type. In addition, CAX3 expression was negatively correlated with the shoot Mg concentration. Together, these results indicate that CAX3 transcription is a quantitative marker of the Mg status in Arabidopsis

Growth Performance and Root Transcriptome Remodeling of Arabidopsis in Response to Mars-Like Levels of Magnesium Sulfate

Martian regolith (unconsolidated surface material) is a potential medium for plant growth in bioregenerative life support systems during manned missions on Mars. However, hydrated magnesium sulfate mineral levels in the regolith of Mars can reach as high as 10 wt%, and would be expected to be highly inhibitory to plant growth. at 180 min. after initiation of treatment. mutants exhibit partial tolerance to magnesium sulfate, and by elucidating a small subset (500 vs. >10,000) of candidate genes for mutation or metabolic engineering that will enhance tolerance to magnesium sulfate soils

Nuclear localised more sulphur accumulation1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over- accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation

Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability

The impact of sulphur limitation on the remobilization of endogenous S compounds during the rosette stage of oilseed rape, and the interactions with N availability on these processes, were examined using a long-term 34SO42− labelling method combined with a study of leaf senescence progression (using SAG12/Cab as a molecular indicator) and gene expression of the transporters, BnSultr4;1 and BnSultr4;2, involved in vacuolar sulphate efflux. After 51 d on hydroponic culture at 0.3 mM 34SO42− (1 atom% excess), the labelling was stopped and plants were subject for 28 d to High S-High N (HS-HN, control), Low S-High N (LS-HN) or Low S-Low N (LS-LN) conditions. Compared with the control, LS-HN plants showed delayed leaf senescence and, whilst the shoot growth and the foliar soluble protein amounts were not affected, S, 34S, and SO42− amounts in the old leaves declined rapidly and were associated with the up-regulation of BnSultr4;1. In LS-LN plants, shoot growth was reduced, leaf senescence was accelerated, and the rapid S mobilization in old leaves was accompanied by decreased 34S and SO42−, higher protein mobilization, and up-regulation of BnSultr4;2, but without any change of expression of BnSultr4;1. The data suggest that to sustain the S demand for growth under S restriction (i) vacuolar SO42− is specifically remobilized in LS-HN conditions without any acceleration of leaf senescence, (ii) SO42− mobilization is related to an up-regulation of BnSultr4;1 and/or BnSultr4;2 expression, and (iii) the relationship between sulphate mobilization and up-regulation of expression of BnSultr4 genes is specifically dependent on the N availability

Effect of mineral sulphur availability on nitrogen and sulphur uptake and remobilization during the vegetative growth of Brassica napus L.

Because it has a high demand for sulphur (S), oilseed rape is particularly sensitive to S limitation. However, the physiological effects of S limitation remain unclear, especially during the rosette stage. For this reason a study was conducted to determine the effects of mineral S limitation on nitrogen (N) and S uptake and remobilization during vegetative growth of oilseed rape at both the whole-plant and leaf rank level for plants grown during 35 d with 300 μM 34SO42– (control plants; +S) or with 15 μM 34SO42– (S-limited plants; –S). The results highlight that S-limited plants showed no significant differences either in whole-plant and leaf biomas or in N uptake, when compared with control plants. However, total S and 34S (i.e. deriving from S uptake) contents were greatly reduced for the whole plant and leaf after 35 d, and a greater redistribution of endogenous S from leaves to the benefit of roots was observed. The relative expression of tonoplast and plasmalemma sulphate transporters was also strongly induced in the roots. In conclusion, although S-limited plants had 20 times less mineral S than control plants, their development remained surprisingly unchanged. During S limitation, oilseed rape is able to recycle endogenous S compounds (mostly sulphate) from leaves to roots. However, this physiological adaptation may be effective only over a short time scale (i.e. vegetative growth)