Functional Analysis of the D- and E- subunits of photosystem I in Arabidopsis thaliana

Although photosynthesis has been intensively studied, many open questions remain, which still need to be answered. The aim of this thesis was to further investigate the PSI complex, which in the course of the light reaction of photosynthesis catalyzes the light-induced transfer of electrons from plastocyanin on the lumenal side to ferredoxin on the stromal side. In this thesis emphasis was put on the reducing side of PSI, so-called stromal ridge of PSI, which is composed of the subunits D, E and C. Functional analyses of Arabidopsis plants carrying disrupted genes for PSI-E and PSI-D subunits were performed. Analyzing PSI-D, it was shown that of the two genes coding for this subunit only a mutation in PSI-D1 led to a general alteration in the polypeptide composition of PSI and thus also in the photosynthetic electron flow. The characterization of psad1-1 psad2-1 double mutant indicated that PSI-D is necessary for the stability of PSI in Arabidopsis. A complete lack of the D subunit led to seedling lethality under photoautotrophic conditions. The instability of Arabidopsis PSI without PSI-D can be explained either by an increase in degradation of the incomplete PSI complex or by downregulation of the synthesis of PSI subunits. In contrast, Arabidopsis plants lacking the PSI-E subunit were able to grow under photoautotrophic conditions. However, they showed severe phenotype including a significant reduction in size and pale green pigmentation, which was turning more yellowish during development. The psae1-3 psae2-1 double mutant exhibited a high-chlorophyll fluorescence phenotype, which had already been observed in the psad1-1 psad2-1 double mutant. This indicates that photosynthetic electron flow is severely altered also in Arabidopsis plants lacking PSI-E subunit. Further spectroscopic, biochemical and physiological studies are in progress in order to understand the biological consequences of a complete lack of PSI-E subunit and its importance for photosynthesis in plants. An unexpected feature observed in both psad1-1 and psae1-3 single mutants, was a significantly increased level of thylakoid protein phosphorylation and therefore the presence of some new phosphopeptides that could not be detected in WT. A striking feature was that even the level of PSI phosphorylation was affected by these mutations. The only PSI phosphopeptide detected so far had been the PSI-D1 protein. Regarding the complementation analysis performed in this thesis it seems that phosphorylation of PSI-D1 does not play a key function in the PSI. Anyhow, it can not be excluded that this phosphorylation might play a role, if plants are grown under particular environmental conditions, not tested in this work. Mass spectrometry techniques and methods of proteomics allowed the successful identification and analysis of one previously unknown phosphoprotein Lhca4. This was the first light-harvesting protein outside of LHCII to be phosphorylated. However, a distinct role for Lhca4 phosphorylation remains unknown. Further studies are needed in order to elucidate the cause and consequences of Lhca4 phosphorylation and to get further insight into the implication of a high level of thylakoid protein phosphorylation as observed in psad1-1 and psae1-3 mutants

The influence of fie and met1 mutations and in vitro culture conditions on autonomous endosperm development in unfertilized ovules of Arabidopsis thaliana

In flowering plants, seeds are produced both sexually (double fertilization is required) and asexually via apomixis (meiotic reduction and egg fertilization are omitted). An apomictic-like pattern of endosperm development in planta is followed by fis mutants of sexual Arabidopsis thaliana. In our experiments in planta, autonomous endosperm (AE) developed in met1 mutants. Furthermore we obtained autonomous endosperm formation in vitro not only in unfertilized ovules of fie mutants but also in wild genotypes (Col-0, MET1/MET1, FIE/FIE) and met1 mutants. AE induction and development occurred in all genotypes on the each of the media used and in every trial. The frequency of AE was relatively high (51.2% ovaries) and genotype-dependent. AE induced in vitro represents a more advanced stage of development than AE induced in fie mutants in planta. This was manifested by a high number of nuclei surrounded by cytoplasm and organized in nuclear cytoplasmic domains (NCDs), nodule formation, division into characteristic regions, and cellularization. The high frequency of AE observed in homozygous met1 (met1/met1) mutants probably is due to accumulation of hypomethylation as an effect of the met1 mutation and the in vitro conditions. AE development was most advanced in FIE/fie mutants. We suggest that changes in the methylation of one or several genes in the DNA of Arabidopsis genotypes caused by in vitro conditions resulted in AE induction and/or further AE development

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

cis‐prenyltransferase 3 and α/β‐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 posttranslational 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/β‐hydrolase—is experimentally confirmed. These data will help to generate Dol‐enriched plants which might serve as a remedy for Dol‐deficiency in humans

The complete chloroplast genome sequence of Platanthera chlorantha (Orchidaceae)

International audienceHere, 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

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 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: A novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis

International audienceIron 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

Arabidopsis STN7 Kinase Provides a Link between Short- and Long-Term Photosynthetic Acclimation[W]

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