Sanja Roje

S-Methylmethionine Plays a Major Role in Phloem Sulfur Transport and Is Synthesized by a Novel Type of Methyltransferase

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem (∼2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l–35S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain (∼300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5′-phosphate–dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Isolation, Characterization, and Functional Expression of cDNAs Encoding NADH-dependent Methylenetetrahydrofolate Reductase from Higher Plants

Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate thatArabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to oneArabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of ≈66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited byS-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10−3. Consistent with this, leaf tissues metabolized [methyl-14C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.

Flavin Nucleotide Metabolism in Plants: MONOFUNCTIONAL ENZYMES SYNTHESIZE FAD IN PLASTIDS*

FAD synthetases (EC 2.7.7.2) catalyze biosynthesis of FAD from FMN and ATP. Monofunctional FAD synthetases are known to exist in mammals and yeast; bifunctional enzymes also catalyzing phosphorylation of riboflavin to FMN are known to exist in bacteria. Previously known eukaryotic enzymes with FAD synthetase activity have no sequence similarity to prokaryotic enzymes with riboflavin kinase and FAD synthetase activities. Proteins homologous to bacterial bifunctional FAD synthetases, yet shorter and lacking amino acid motifs at the C terminus, were found by bioinformatic analyses in vascular plant genomes, suggesting that plants contain a type of FAD synthetase previously known to exist only in prokaryotes. The Arabidopsis thaliana genome encodes two of such proteins. Both proteins, which we named AtRibF1 and AtRibF2, carry N-terminal extensions with characteristics of organellar targeting peptides. AtRibF1 and AtRibF2 cDNAs were cloned by reverse transcription-PCR. Only FAD synthetase activity was detected in the recombinant enzymes produced in Escherichia coli. FMN and ATP inhibited both enzymes. Kinetic parameters of AtRibF1 and AtRibF2 for the two substrates were similar. Confocal microscopy of protoplasts transformed with enhanced green fluorescence protein-fused proteins showed that AtRibF1 and AtRibF2 are targeted to plastids. In agreement with subcellular localization to plastids, Percoll-isolated chloroplasts from pea (Pisum sativum) synthesized FAD from imported riboflavin. Riboflavin kinase, FMN hydrolase, and FAD pyrophosphatase activities were detected in Percoll-isolated chloroplasts and mitochondria from pea. We propose from these new findings a model for subcellular distribution of enzymes that synthesize and hydrolyze flavin nucleotides in plants.

A dual regulatory role of Arabidopsis calreticulin-2 in plant innate immunity.

Calreticulin (CRT) is an endoplasmic reticulum-resident calcium-binding molecular chaperone that is highly conserved in multi-cellular eukaryotes. Higher plants contain two distinct groups of CRTs: CRT1/CRT2 and CRT3 isoforms. Previous studies have shown that bacterial elongation factor Tu receptor (EFR), a pattern-recognition receptor that is responsible for pathogen-associated molecular pattern-triggered immunity, is a substrate for Arabidopsis CRT3, suggesting a role for CRT3 in regulating plant defense against pathogens. Here we report that Arabidopsis CRT2 is another regulator of plant innate immunity. Despite significantly increased salicylic acid levels and constitutive expression of the systemic acquired resistance-associated marker genes PR1, PR2 and PR5, transgenic plants over-expressing CRT2 displayed reduced resistance to virulent Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A (45)Ca(2+) overlay assay and a domain-swapping experiment further demonstrated that the negatively charged C-terminal tail of CRT2 is responsible for its high calcium-binding capacity and function in regulating the endogenous salicylic acid level. In addition, over-expression of the His173 mutant of CRT2 greatly enhanced plant defense against PstDC3000, supporting the existence of a self-inhibition mechanism that can counteract the effects of salicylic acid-dependent immune responses. These results suggest that CRT2 functions through its N-terminal domain(s) as a self-modulator that can possibly prevent the salicylic acid-mediated runaway defense responses triggered by its C-terminal calcium-buffering activity in response to pathogen invasion.

An FMN Hydrolase Is Fused to a Riboflavin Kinase Homolog in Plants

Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg2+ in the assays, as expected for a member of the Mg2+-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties.

Repression of Sulfate Assimilation Is an Adaptive Response of Yeast to the Oxidative Stress of Zinc Deficiency

The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. Previous studies identified over 80 genes activated by Zap1 in zinc-limited cells. In this report, we identified 36 genes repressed in a zinc- and Zap1-responsive manner. As a result, we have identified a new mechanism of Zap1-mediated gene repression whereby transcription of the MET3, MET14, and MET16 genes is repressed in zinc-limited cells. These genes encode the first three enzymes of the sulfate assimilation pathway. We found that MET30, encoding a component of the SCFMet30 ubiquitin ligase, is a direct Zap1 target gene. MET30 expression is increased in zinc-limited cells, and this leads to degradation of Met4, a transcription factor responsible for MET3, MET14, and MET16 expression. Thus, Zap1 is responsible for a decrease in sulfate assimilation in zinc-limited cells. We further show that cells that are unable to down-regulate sulfate assimilation under zinc deficiency experience increased oxidative stress. This increased oxidative stress is associated with an increase in the NADP+/NADPH ratio and may result from a decrease in NADPH-dependent antioxidant activities. These studies have led to new insights into how cells adapt to nutrient-limiting growth conditions.

Perspectives on plant photorespiratory metabolism

Being intimately intertwined with (C3) photosynthesis, photorespiration is an incredibly high flux-bearing pathway. Traditionally, the photorespiratory cycle was viewed as closed pathway to refill the Calvin-Benson cycle with organic carbon. However, given the network nature of metabolism, it hence follows that photorespiration will interact with many other pathways. In this article, we review current understanding of these interactions and attempt to define key priorities for future research, which will allow us greater fundamental comprehension of general metabolic and developmental consequences of perturbation of this crucial metabolic process.

Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants

Plants regulate transcriptional processes to coordinate protein synthesis in response to environmental cues. Here it is shown that CULLIN3-based E3 ubiquitin ligases, which help mark proteins for degradation via the 26S proteasome, are key regulators of processes involving ERF/AP2 transcription factors via the activity of MATH-BTB/POZ proteins, which affect metabolism and fatty acid content. Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

One-carbon metabolism in plants: characterization of a plastid serine hydroxymethyltransferase

SHMT (serine hydroxymethyltransferase; EC 2.1.2.1) catalyses reversible hydroxymethyl group transfer from serine to H4PteGlun (tetrahydrofolate), yielding glycine and 5,10-methylenetetrahydrofolate. In plastids, SHMTs are thought to catalytically direct the hydroxymethyl moiety of serine into the metabolic network of H4PteGlun-bound one-carbon units. Genes encoding putative plastid SHMTs were found in the genomes of various plant species. SHMT activity was detected in chloroplasts in pea (Pisum sativum) and barley (Hordeum vulgare), suggesting that plastid SHMTs exist in all flowering plants. The Arabidopsis thaliana genome encodes one putative plastid SHMT (AtSHMT3). Its cDNA was cloned by reverse transcription-PCR and the encoded recombinant protein was produced in Escherichia coli. Evidence that AtSHMT3 is targeted to plastids was found by confocal microscopy of A. thaliana protoplasts transformed with proteins fused to enhanced green fluorescent protein. Characterization of recombinant AtSHMT3 revealed that substrate affinity for and the catalytic efficiency of H4PteGlu1-8 increase with n, and that H4PteGlu1-8 inhibit AtSHMT3. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate with one and five glutamate residues inhibited AtSHMT3-catalysed hydroxymethyl group transfer from serine to H4PteGlu6, with the pentaglutamylated inhibitors being more effective. Calculations revealed inhibition with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate resulting in little reduction in AtSHMT3 activity under folate concentrations estimated for plastids.

Folate Biosynthesis in Higher Plants. cDNA Cloning, Heterologous Expression, and Characterization of Dihydroneopterin Aldolases

Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1–3, sharing 57%–73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.