cis-prenyltransferase 3 and α/ß-hydrolase are new determinants of dolichol accumulation in Arabidopsis.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for post-translational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis-the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/ß-hydrolase-is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Interplay between Coumarin Accumulation, Iron Deficiency and Plant Resistance to Dickeya spp.

Coumarins belong to a group of secondary metabolites well known for their high biological activities including antibacterial and antifungal properties. Recently, an important role of coumarins in plant resistance to pathogens and their release into the rhizosphere upon pathogen infection was discovered. It is also well documented that coumarins play a crucial role in the Arabidopsis thaliana growth under Fe-limited conditions. However, the mechanisms underlying interplay between plant resistance, accumulation of coumarins and Fe status, remain largely unknown. In this work, we investigated the effect of both mentioned factors on the disease severity using the model system of Arabidopsis/Dickeya spp. molecular interactions. We evaluated the disease symptoms in Arabidopsis plants, wild-type Col-0 and its mutants defective in coumarin accumulation, grown in hydroponic cultures with contrasting Fe regimes and in soil mixes. Under all tested conditions, Arabidopsis plants inoculated with Dickeya solani IFB0099 strain developed more severe disease symptoms compared to lines inoculated with Dickeya dadantii 3937. We also showed that the expression of genes encoding plant stress markers were strongly affected by D. solani IFB0099 infection. Interestingly, the response of plants to D. dadantii 3937 infection was genotype-dependent in Fe-deficient hydroponic solution.

Identification and Quantification of Coumarins by UHPLC-MS in Arabidopsis thaliana Natural Populations.

Coumarins are phytochemicals occurring in the plant kingdom, which biosynthesis is induced under various stress factors. They belong to the wide class of specialized metabolites well known for their beneficial properties. Due to their high and wide biological activities, coumarins are important not only for the survival of plants in changing environmental conditions, but are of great importance in the pharmaceutical industry and are an active source for drug development. The identification of coumarins from natural sources has been reported for different plant species including a model plant Arabidopsis thaliana. In our previous work, we demonstrated a presence of naturally occurring intraspecies variation in the concentrations of scopoletin and its glycoside, scopolin, the major coumarins accumulating in Arabidopsis roots. Here, we expanded this work by examining a larger group of 28 Arabidopsis natural populations (called accessions) and by extracting and analysing coumarins from two different types of tissues-roots and leaves. In the current work, by quantifying the coumarin content in plant extracts with ultra-high-performance liquid chromatography coupled with a mass spectrometry analysis (UHPLC-MS), we detected a significant natural variation in the content of simple coumarins like scopoletin, umbelliferone and esculetin together with their glycosides: scopolin, skimmin and esculin, respectively. Increasing our knowledge of coumarin accumulation in Arabidopsis natural populations, might be beneficial for the future discovery of physiological mechanisms of action of various alleles involved in their biosynthesis. A better understanding of biosynthetic pathways of biologically active compounds is the prerequisite step in undertaking a metabolic engineering research.

Scopoletin 8-hydroxylase: a novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis.

Iron deficiency is a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under iron deficiency. An essential enzyme for the biosynthesis of the major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6'-Hydroxylase1 (F6'H1), which belongs to a large enzyme family of the 2-oxoglutarate and Fe2+-dependent dioxygenases. We have functionally characterized another enzyme of this family, which is a close homologue of F6'H1 and is encoded by a strongly iron-responsive gene, At3g12900. We purified At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin, via hydroxylation at the C8 position, and that it thus functions as a scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves, followed by metabolite profiling and biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various iron regimes. Our results indicate that S8H is involved in coumarin biosynthesis, as part of mechanisms used by plants to assimilate iron.

Natural variation for anthocyanin accumulation under high-light and low-temperature stress is attributable to the ENHANCER OF AG-4 2 (HUA2) locus in combination with PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2.

Growing conditions combining high light intensities and low temperatures lead to anthocyanin accumulation in plants. This response was contrasted between two Arabidopsis thaliana accessions, which were used to decipher the genetic and molecular bases underlying the variation of this response. Quantitative trait loci (QTLs) for flowering time (FT) and anthocyanin accumulation under a high-light and low-temperature scenario versus a control environment were mapped. Major QTLs were confirmed using near-isogenic lines. Candidate genes were examined using mutants and gene expression studies as well as transgenic complementation. Several QTLs were found for FT and for anthocyanin content, of which one QTL co-located at the ENHANCER OF AG-4 2 (HUA2) locus. That HUA2 is a regulator of both pathways was confirmed by the analysis of loss-of-function mutants. For a strong expression of anthocyanin, additional allelic variation was detected for the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2 genes which control the anthocyanin pathway. The genetic control of variation for anthocyanin content was dissected in A. thaliana and shown to be affected by a common regulator of flowering and anthocyanin biosynthesis together with anthocyanin-specific regulators.

Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana.

BACKGROUND: Scopoletin and its glucoside scopolin are important secondary metabolites synthesized in plants as a defense mechanism against various environmental stresses. They belong to coumarins, a class of phytochemicals with significant biological activities that is widely used in medical application and cosmetics industry. Although numerous studies showed that a variety of coumarins occurs naturally in several plant species, the details of coumarins biosynthesis and its regulation is not well understood. It was shown previously that coumarins (predominantly scopolin and scopoletin) occur in Arabidopsis thaliana (Arabidopsis) roots, but until now nothing is known about natural variation of their accumulation in this model plant. Therefore, the genetic architecture of coumarins biosynthesis in Arabidopsis has not been studied before. RESULTS: Here, the variation in scopolin and scopoletin content was assessed by comparing seven Arabidopsis accessions. Subsequently, a quantitative trait locus (QTL) mapping was performed with an Advanced Intercross Recombinant Inbred Lines (AI-RILs) mapping population EstC (Est-1 × Col). In order to reveal the genetic basis of both scopolin and scopoletin biosynthesis, two sets of methanol extracts were made from Arabidopsis roots and one set was additionally subjected to enzymatic hydrolysis prior to quantification done by high-performance liquid chromatography (HPLC). We identified one QTL for scopolin and five QTLs for scopoletin accumulation. The identified QTLs explained 13.86% and 37.60% of the observed phenotypic variation in scopolin and scopoletin content, respectively. In silico analysis of genes located in the associated QTL intervals identified a number of possible candidate genes involved in coumarins biosynthesis. CONCLUSIONS: Together, our results demonstrate for the first time that Arabidopsis is an excellent model for studying the genetic and molecular basis of natural variation in coumarins biosynthesis in plants. It additionally provides a basis for fine mapping and cloning of the genes involved in scopolin and scopoletin biosynthesis. Importantly, we have identified new loci for this biosynthetic process.

Conserved histidine of metal transporter AtNRAMP1 is crucial for optimal plant growth under manganese deficiency at chilling temperatures.

Manganese (Mn) is an essential nutrient required for plant growth, in particular in the process of photosynthesis. Plant performance is influenced by various environmental stresses including contrasting temperatures, light or nutrient deficiencies. The molecular responses of plants exposed to such stress factors in combination are largely unknown. Screening of 108 Arabidopsis thaliana (Arabidopsis) accessions for reduced photosynthetic performance at chilling temperatures was performed and one accession (Hog) was isolated. Using genetic and molecular approaches, the molecular basis of this particular response to temperature (G × E interaction) was identified. Hog showed an induction of a severe leaf chlorosis and impaired growth after transfer to lower temperatures. We demonstrated that this response was dependent on the nutrient content of the soil. Genetic mapping and complementation identified NRAMP1 as the causal gene. Chlorotic phenotype was associated with a histidine to tyrosine (H239Y) substitution in the allele of Hog NRAMP1. This led to lethality when Hog seedlings were directly grown at 4°C. Chemical complementation and hydroponic culture experiments showed that Mn deficiency was the major cause of this G × E interaction. For the first time, the NRAMP-specific highly conserved histidine was shown to be crucial for plant performance.

The complete chloroplast genome sequence of Platanthera chlorantha (Orchidaceae).

Here, we report the first complete chloroplast genome of Platanthera chlorantha (Orchidaceae: Orchidoideae). The circular genome with the length of 154,260 bp possesses the typical structure consisting of a large single copy region (LSC) of 83,279 bp and a small single copy region (SSC) of 17,759 bp, separated from each other by two copies of inverted repeats (IRs) of 26,611 bp. The plastome encodes 134 genes, of which 88 were protein-coding, eight encoded ribosomal RNA, and 38 transfer RNAs. The overall GC content was 36.74%. The plastome sequence provided here constitutes a valuable resource for analyzing genetic diversity of the Orchidaceae family.

Arabidopsis STN7 kinase provides a link between short- and long-term photosynthetic acclimation.

Flowering plants control energy allocation to their photosystems in response to light quality changes. This includes the phosphorylation and migration of light-harvesting complex II (LHCII) proteins (state transitions or short-term response) as well as long-term alterations in thylakoid composition (long-term response or LTR). Both responses require the thylakoid protein kinase STN7. Here, we show that the signaling pathways triggering state transitions and LTR diverge at, or immediately downstream from, STN7. Both responses require STN7 activity that can be regulated according to the plastoquinone pool redox state. However, LTR signaling does not involve LHCII phosphorylation or any other state transition step. State transitions appear to play a prominent role in flowering plants, and the ability to perform state transitions becomes critical for photosynthesis in Arabidopsis thaliana mutants that are impaired in thylakoid electron transport but retain a functional LTR. Our data imply that STN7-dependent phosphorylation of an as yet unknown thylakoid protein triggers LTR signaling events, whereby an involvement of the TSP9 protein in the signaling pathway could be excluded. The LTR signaling events then ultimately regulate in chloroplasts the expression of photosynthesis-related genes on the transcript level, whereas expression of nuclear-encoded proteins is regulated at multiple levels, as indicated by transcript and protein profiling in LTR mutants.

Impaired photosystem I oxidation induces STN7-dependent phosphorylation of the light-harvesting complex I protein Lhca4 in Arabidopsis thaliana.

Reduction of the plastoquinone (PQ) pool is known to activate phosphorylation of thylakoid proteins. In the Arabidopsis thaliana mutants psad1-1 and psae1-3, oxidation of photosystem I (PSI) is impaired, and the PQ pool is correspondingly over-reduced. We show here that, under these conditions, the antenna protein Lhca4 of PSI becomes a target for phosphorylation. Phosphorylation of the mature Lhca4 protein at Thr16 is suppressed in stn7 psad1 and stn7 psae1 double mutants. Thus, under extreme redox conditions, hyperactivation of thylakoid protein kinases and/or reorganization of thylakoid protein complex distribution increase the susceptibility of PSI to phosphorylation.

The E subunit of photosystem I is not essential for linear electron flow and photoautotrophic growth in Arabidopsis thaliana.

PSI-E is part of the stromal side of photosystem I (PSI). In Arabidopsis thaliana, the two nuclear genes PsaE1 and PsaE2 code for PSI-E, and transcripts of PsaE1 are markedly more abundant than PsaE2 transcripts. Stable null alleles of the two PsaE genes, psae1-3 and psae2-1, were identified and characterised. The psae2-1 mutant exhibited wild-type like PSI-E abundance and photosynthetic performance, whereas in the psae1-3 mutant PSI-E accumulation was decreased by 85%, together with an impaired thylakoid electron flow and plant growth rate. The psae1-3 psae2-1 double mutant totally lacked PSI-E but was still able to grow photoautotrophically, implying that PSI-E is not essential for PSI accumulation and thylakoid electron flow.

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions.

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.