Search

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (−S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under –S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency., Sulfur Deficiency Induced1 (SDI1) upregulates S-poor seed storage proteins in favor of S-rich seed storage proteins.

The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis (Arabidopsis thaliana) mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.

The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco (Nicotiana tabacum) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis.

Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs. Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c. Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c. Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs. Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves.

2',3'-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2',3'-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2',3'-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2',3'-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2',3'-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2',3'-cAMP. Furthermore, SG formation was promoted in 2',3'-cAMP-treated Arabidopsis seedlings, and interactions between 2',3'-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2',3'-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.

A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43′ protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.