Michael A. Phillips

Distinct Light-Mediated Pathways Regulate the Biosynthesis and Exchange of Isoprenoid Precursors during Arabidopsis Seedling Development

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.

The plastidial MEP pathway: unified nomenclature and resources

In plants, the plastid-localized 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway provides the precursors for the synthesis of isoprenoid hormones, monoterpenes, carotenoids and the side chain of chlorophylls, tocopherols and prenylquinones. As a result of the fast progress in the elucidation and characterization of the pathway (mainly by genetic approaches in Escherichia coli and Arabidopsis thaliana), different names have been used in the literature to designate the orthologous bacterial and plant genes and the corresponding null and partial loss-of-function mutants. This has led to a confusing variety of naming conventions in this field. Here, we propose a reorganization of the various naming systems with the aim of facilitating the dissemination and sharing of genetic resources and tools central to plant isoprenoid research.

Functional identification and differential expression of 1-deoxy-D-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies).

Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.

The Arabidopsis thaliana Type I Isopentenyl Diphosphate Isomerases Are Targeted to Multiple Subcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

To form the building blocks of isoprenoids, isopentenyl diphosphate (IPP) isomerase activity, which converts IPP to dimethylallyl diphosphate (DMAPP), appears to be necessary in cytosol, plastids, and mitochondria. Arabidopsis thaliana contains only two IPP isomerases (Isopentenyl Diphosphate Isomerase1 [IDI1] and IDI2). Both encode proteins with N-terminal extensions similar to transit peptides and are expressed in all organs, with IDI1 less abundant than IDI2. Examination of enhanced green fluorescent protein fusions established that IDI1 is mainly in the plastid, whereas IDI2 is mainly in the mitochondria. Both proteins are also in the cytosol as a result of their translation from naturally occurring shorter transcripts lacking transit peptides, as demonstrated by 5′ rapid amplification of cDNA ends cloning. IPP isomerase activity in the cytosol was confirmed by uniform labeling of IPP- and DMAPP-derived units of the cytoplasmic isoprenoid product, sitosterol, when labeled mevalonate was administered. Analysis of mutant lines showed that double mutants were nonviable, while homozygous single mutants had no major morphological or chemical differences from the wild type except for flowers with fused sepals and underdeveloped petals on idi2 mutants. Thus, each of the two Arabidopsis IPP isomerases is found in multiple but partially overlapping subcellular locations, and each can compensate for the loss of the other through partial redundancy in the cytosol.

Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis

1-Deoxyxylulose 5-phosphate synthase is the controlling enzyme of plastid isoprenoid precursor biosynthesis in Arabidopsis. The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) Integrates the Regulation of Sugar Responses with Isoprenoid Metabolism in Arabidopsis

The biosynthesis of isoprenoids in plant cells occurs from precursors produced in the cytosol by the mevalonate (MVA) pathway and in the plastid by the methylerythritol 4-phosphate (MEP) pathway, but little is known about the mechanisms coordinating both pathways. Evidence of the importance of sugar signaling for such coordination in Arabidopsis thaliana is provided here by the characterization of a mutant showing an increased accumulation of MEP-derived isoprenoid products (chlorophylls and carotenoids) without changes in the levels of relevant MEP pathway transcripts, proteins, or enzyme activities. This mutant was found to be a new loss-of-function allele of PRL1 (Pleiotropic Regulatory Locus 1), a gene encoding a conserved WD-protein that functions as a global regulator of sugar, stress, and hormone responses, in part by inhibition of SNF1-related protein kinases (SnRK1). Consistent with the reported role of SnRK1 kinases in the phosphorylation and inactivation of the main regulatory enzyme of the MVA pathway (hydroxymethylglutaryl coenzyme-A reductase), its activity but not transcript or protein levels was reduced in prl1 seedlings. However, the accumulation of MVA-derived end products (sterols) was unaltered in mutant seedlings. Sucrose supplementation to wild-type seedlings phenocopied the prl1 mutation in terms of isoprenoid metabolism, suggesting that the observed isoprenoid phenotypes result from the increased sugar accumulation in the prl1 mutant. In summary, PRL1 appears to coordinate isoprenoid metabolism with sugar, hormone, and stress responses.

Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control

Protein quality control mechanisms rely on chaperones and proteases to maintain cell proteins in working conditions. This study reports the identification of a J-protein cochaperone that binds to inactive forms of a plastidial enzyme required for plant photosynthesis and development, targeting them for either proper folding or degradation in the chloroplast. Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.

Evaluation of Candidate Reference Genes for Real-Time Quantitative PCR of Plant Samples Using Purified cDNA as Template

Quantitative real-time polymerase chain reaction (qRT-PCR) is a precise method to measure changes in gene transcript level. Accurate quantification requires careful RNA quality assessment, determination of primer efficiency, and selection of an appropriate reference gene. While many experimental procedures for these purposes have been described for mammalian samples, the direct application of these methods to plant samples often introduces unexpected experimental errors due to the complex and variable nature of the ribosomal RNA species present in typical plant extracts. In this paper, we report a simple procedure for the purification and quantification of complementary DNA (cDNA) after reverse transcriptase reactions by microcapillary electrophoresis. The use of purified cDNA allows template concentrations to be more accurately standardized for SYBR Green PCR reactions and increases amplification efficiencies so that these closely resemble those determined by the standard curve method. These advantages facilitate a more precise evaluation of the transcript levels of candidate reference genes under various experimental conditions without bias from differences in reverse transcriptase efficiency, template loading, or the presence of PCR inhibitors following reverse transcription. Using samples from Arabidopsis thaliana and Picea abies (Norway spruce), we demonstrate the value of this approach for selecting reference genes.

Quantitative iTRAQ proteome and comparative transcriptome analysis of elicitor-induced Norway spruce (Picea abies) cells reveals elements of calcium signaling in the early conifer defense response

Long‐lived conifer trees depend on both constitutive and induced defenses for resistance against a myriad of potential pathogens and herbivores. In species of spruce (Picea spp.), several of the late events of pathogen‐, insect‐, or elicitor‐induced defense responses have previously been characterized at the anatomical, biochemical, transcriptome, and proteome levels in stems and needles. However, accurately measuring the early events of induced cellular responses in a conifer is technically challenging due to limitations in the precise timing of induction and tissue sampling from intact trees following insect or fungal treatment. In the present study, we used the advantages of Norway spruce (Picea abies) cell suspensions combined with chitosan elicitation to investigate the early proteome response in a conifer. A combination of iTRAQ labeling and a new design of iterative sample analysis employing data‐dependent exclusion lists were used for proteome analysis. This approach improved the coverage of the spruce proteome beyond that achieved in any prior study in a conifer system. Comparison of elicitor‐induced proteome and transcriptome responses in Norway spruce cells consistently identified features associated with calcium‐mediated signaling and response to oxidative stress that have not previously been observed in the response of intact trees to fungal attack.