PSB33 protein sustains photosystem II in plant chloroplasts under UV-A light.

Plants can quickly and dynamically respond to spectral and intensity variations of the incident light. These responses include activation of developmental processes, morphological changes, and photosynthetic acclimation that ensure optimal energy conversion and minimal photoinhibition. Plant adaptation and acclimation to environmental changes have been extensively studied, but many details surrounding these processes remain elusive. The photosystem II (PSII)-associated protein PSB33 plays a fundamental role in sustaining PSII as well as in the regulation of the light antenna in fluctuating light. We investigated how PSB33 knock-out Arabidopsis plants perform under different light qualities. psb33 plants displayed a reduction of 88% of total fresh weight compared to wild type plants when cultivated at the boundary of UV-A and blue light. The sensitivity towards UV-A light was associated with a lower abundance of PSII proteins, which reduces psb33 plants' capacity for photosynthesis. The UV-A phenotype was found to be linked to altered phytohormone status and changed thylakoid ultrastructure. Our results collectively show that PSB33 is involved in a UV-A light-mediated mechanism to maintain a functional PSII pool in the chloroplast.

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.

Involvement of the electrophilic isothiocyanate sulforaphane in Arabidopsis local defense responses.

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue.

The activity of HYDROPEROXIDE LYASE 1 regulates accumulation of galactolipids containing 12-oxo-phytodienoic acid in Arabidopsis.

Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids.

Role of the penetration-resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race-specific resistance in Arabidopsis thaliana.

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host-encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall-based defense against the non-adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run-away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall-based defense against non-host pathogens.

Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in Arabidopsis thaliana.

Non-specific lipid transfer proteins (LTPs) are involved in the transport of lipophilic compounds to the cuticular surface in epidermal cells and in the defence against pathogens. The role of glycophosphatidylinositol (GPI)-anchored LTPs (LTPGs) in resistance against non-host mildews in Arabidopsis thaliana was investigated using reverse genetics. Loss of either LTPG1, LTPG2, LTPG5 or LTPG6 increased the susceptibility to penetration of the epidermal cell wall by Blumeria graminis f. sp. hordei (Bgh). However, no impact on pre-penetration defence against another non-host mildew, Erysiphe pisi (Ep), was observed. LTPG1 was localized to papillae at the sites of Bgh penetration. This study shows that, in addition to the previously known functions, LTPGs contribute to pre-invasive defence against certain non-host powdery mildew pathogens.

Complete Genome Sequence of Novel Sulfitobacter pseudonitzschiae Strain SMR1, Isolated from a Culture of the Marine Diatom Skeletonema marinoi.

When studying diatoms, an important consideration is the role of associated bacteria in the diatom-microbiome holobiont. To that end, bacteria isolated from a culture of Skeletonema marinoi strain R05AC were sequenced, one of which being bacterial strain SMR1, presented here. The genome consists of a circular chromosome and seven circular plasmids, totalling 5,121,602 bp. After phylotaxonomic analysis and 16S rRNA sequence comparison, we place this strain in the taxon Sulfitobacter pseudonitzschiae on account of similarity to the type strain. The annotated genome suggests similar interactions between strain SMR1 and its host diatom as have been shown previously in diatom-associated Sulfitobacter, for example bacterial production of growth hormone for its host, and breakdown of diatom-derived DMSP by Sulfitobacter for use as a sulfur source.

Genome Sequence of Arenibacter algicola Strain SMS7, Found in Association with the Marine Diatom Skeletonema marinoi.

Arenibacter algicola strain SMS7 was isolated from a culture of the marine diatom Skeletonema marinoi strain ST54, sampled from top-layer sediments in Kosterfjord, Sweden. Here, we present its 5,857,781-bp genome, consisting of a circular chromosome and one circular plasmid, in all containing 4,932 coding sequences.

Skeletonema marinoi as a new genetic model for marine chain-forming diatoms.

Diatoms are ubiquitous primary producers in marine ecosystems and freshwater habitats. Due to their complex evolutionary history, much remains unknown about the specific gene functions in diatoms that underlie their broad ecological success. In this study, we have genetically transformed the centric diatom Skeletonema marinoi, a dominant phytoplankton species in temperate coastal regions. Transformation of S. marinoi is the first for a true chain-forming diatom, with the random genomic integration via nonhomologous recombination of a linear DNA construct expressing the resistance gene to the antibiotic zeocin. A set of molecular tools were developed for reliably identifying the genomic insertion site within each transformant, many of which disrupt recognizable genes and constitute null or knock-down mutations. We now propose S. marinoi as a new genetic model for marine diatoms, representing true chain-forming species that play a central role in global photosynthetic carbon sequestration and the biogeochemical cycling of silicates and various nutrients, as well as having potential biotechnological applications.

Friends With Benefits: Exploring the Phycosphere of the Marine Diatom Skeletonema marinoi.

Marine diatoms are the dominant phytoplankton in the temperate oceans and coastal regions, contributing to global photosynthesis, biogeochemical cycling of key nutrients and minerals and aquatic food chains. Integral to the success of marine diatoms is a diverse array of bacterial species that closely interact within the diffusive boundary layer, or phycosphere, surrounding the diatom partner. Recently, we isolated seven distinct bacterial species from cultures of Skeletonema marinoi, a chain-forming, centric diatom that dominates the coastal regions of the temperate oceans. Genomes of all seven bacteria were sequenced revealing many unusual characteristics such as the existence of numerous plasmids of widely varying sizes. Here we have investigated the characteristics of the bacterial interactions with S. marinoi, demonstrating that several strains (Arenibacter algicola strain SMS7, Marinobacter salarius strain SMR5, Sphingorhabdus flavimaris strain SMR4y, Sulfitobacter pseudonitzschiae strain SMR1, Yoonia vestfoldensis strain SMR4r and Roseovarius mucosus strain SMR3) stimulate growth of the diatom partner. Testing of many different environmental factors including low iron concentration, high and low temperatures, and chemical signals showed variable effects on this growth enhancement by each bacterial species, with the most significant being light quality in which green and blue but not red light enhanced the stimulatory effect on S. marinoi growth by all bacteria. Several of the bacteria also inhibited growth of one or more of the other bacterial strains to different extents when mixed together. This study highlights the complex interactions between diatoms and their associated bacteria within the phycosphere, and that further studies are needed to resolve the underlying mechanisms for these relationships and how they might influence the global success of marine diatoms.

Genome Sequence of Kordia sp. Strain SMS9 Identified in a Non-Axenic Culture of the Diatom Skeletonema marinoi.

Initial efforts to sequence the genome of the marine diatom Skeletonema marinoi were hampered by the presence of genetic material from bacteria, and there was sufficient material from some of these bacteria to enable the assembly of full chromosomes. Here, we report the genome of strain SMS9, one such bacterial species identified in a non-axenic culture of S. marinoi strain ST54. Its 5,482,391 bp circular chromosome contains 4,641 CDSs, and has a G+C content of 35.6%. Based on 16S rRNA comparison, phylotaxonomic analysis, and the genome similarity metrics dDDH and OrthoANI, we place this strain in the genus Kordia, and to the best of our knowledge, this is the first Kordia species to be initially described from European waters. As attempts to culture this strain have failed, however, the specifics of its relationship with S. marinoi are still uncertain.

Complete Genome Sequence of the Diatom-Associated Bacterium Sphingorhabdus sp. Strain SMR4y.

The bacterial strain SMR4y belongs to the diverse microbiome of the marine diatom Skeletonema marinoi strain R05AC. After assembly of its genome, presented here, and subsequent analyses, we placed it in the genus Sphingorhabdus This strain has a 3,479,724-bp circular chromosome (with 3,340 coding sequences) and no known plasmids.

Phenomics reveals a novel putative chloroplast fatty acid transporter in the marine diatom Skeletonema marinoi involved in temperature acclimation.

Diatoms are the dominant phytoplankton in temperate oceans and coastal regions and yet little is known about the genetic basis underpinning their global success. Here, we address this challenge by developing the first phenomic approach for a diatom, screening a collection of randomly mutagenized but identifiably tagged transformants. Based upon their tolerance to temperature extremes, several compromised mutants were identified revealing genes either stress related or encoding hypothetical proteins of unknown function. We reveal one of these hypothetical proteins is a novel putative chloroplast fatty acid transporter whose loss affects several fatty acids including the two omega-3, long-chain polyunsaturated fatty acids - eicosapentaenoic and docosahexaenoic acid, both of which have medical importance as dietary supplements and industrial significance in aquaculture and biofuels. This mutant phenotype not only provides new insights into the fatty acid biosynthetic pathways in diatoms but also highlights the future value of phenomics for revealing specific gene functions in these ecologically important phytoplankton.

Whole Genome Sequence of Marinobacter salarius Strain SMR5, Shown to Promote Growth in its Diatom Host.

Attempts to obtain axenic cultures of the marine diatom Skeletonema marinoi often result in poor growth, indicating the importance of the microbiome to the growth of its host. In order to identify the precise roles played by these associated bacteria, individual strains were isolated, cultured and sequenced. We report the genome of one such strain - SMR5, isolated from a culture of S. marinoi strain R05AC sampled from top layer sediments of the Swedish west coast. Its genome of 4,630,160 bp consists of a circular chromosome and one circular plasmid, and 4,263 CDSs were inferred in the annotation. Comparison of 16S rRNA sequences and other markers, along with phylotaxonomic analysis, leads us to place strain SMR5 in the taxon Marinobacter salarius. Pathway analysis and previous experimental work suggest that this strain may produce a growth factor, as well as improve iron availability for its host via siderophores.

Complete Genome Sequence of Loktanella vestfoldensis Strain SMR4r, a Novel Strain Isolated from a Culture of the Chain-Forming Diatom Skeletonema marinoi.

We report here the genome sequence of Loktanella vestfoldensis strain SMR4r, isolated from the marine diatom Skeletonema marinoi strain RO5AC. Its 3,987,360-bp genome consists of a circular chromosome and two circular plasmids, one of which appears to be shared with an S. marinoi-associated Roseovarius species.

Whole-Genome Sequence of the Novel Antarctobacter heliothermus Strain SMS3, Found in Association with the Marine Diatom Skeletonema marinoi.

As part of an ongoing investigation into the microbiome of the marine diatom Skeletonema marinoi, the bacterial strain SMS3 was isolated from a culture of S. marinoi strain ST54, which had been propagated from a sample of top layer marine sediments taken from the Swedish west coast. We present here the sequenced genome of this bacterium, which we place in the taxon Antarctobacter heliothermus, based on a phylotaxonomic analysis and its high 16S rRNA sequence similarity to the A. heliothermus type strain DSM 11445T. Its 5,331,190 bp genome consists of a circular chromosome and three circular plasmids, and contains 5,019 CDSs. Strain SMS3 contains a phosphatidylcholine synthase gene, as well as genes involved in DMSP degradation, both of which imply a potential symbiotic relationship with its host.

Genome Sequence of Roseovarius mucosus Strain SMR3, Isolated from a Culture of the Diatom Skeletonema marinoi.

We present the genome of Roseovarius mucosus strain SMR3, a marine bacterium isolated from the diatom Skeletonema marinoi strain RO5AC sampled from top layer sediments at 14 m depth. Its 4,381,426 bp genome consists of a circular chromosome and two circular plasmids and contains 4,178 coding sequences (CDSs).

A voltage-dependent chloride channel fine-tunes photosynthesis in plants.

In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl(-)) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl(-) channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.

A quick and robust method for quantification of the hypersensitive response in plants.

One of the most studied defense reactions of plants against microbial pathogens is the hypersensitive response (HR). The HR is a complex multicellular process that involves programmed cell death at the site of infection. A standard method to quantify plant defense and the HR is to measure the release of cellular electrolytes into water after infiltration with pathogenic bacteria. In this type of experiment, the bacteria are typically delivered into the plant tissue through syringe infiltration. Here we report the development of a vacuum infiltration protocol that allows multiple plant lines to be infiltrated simultaneously and assayed for defense responses. Vacuum infiltration did not induce more wounding response in Arabidopsis leaf tissue than syringe inoculation, whereas throughput and reproducibility were improved. The method was used to study HR-induced electrolyte loss after treatment with the bacterium Pseudomonas syringae pv. tomato DC3000 harboring the effector AvrRpm1, AvrRpt2 or AvrRps4. Specifically, the influence of bacterial titer on AvrRpm1-induced HR was investigated. Not only the amplitude, but also the timing of the maximum rate of the HR reaction was found to be dose-dependent. Finally, using vacuum infiltration, we were able quantify induction of phospholipase D activity after AvrRpm1 recognition in leaves labeled with (33)PO4.

Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana.

Plants possess a highly sophisticated system for defense against microorganisms. So called MAMP (microbe-associated molecular patterns) triggered immunity (MTI) prevents the majority of non-adapted pathogens from causing disease. Adapted plant pathogens use secreted effector proteins to interfere with such signaling. Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI). The latter usually comprises the hypersensitive response (HR) which includes programmed cell death at the site of infection. Phospholipase D (PLD) mediated production of phosphatidic acid (PA) has been linked to both MTI and ETI in plants. Inhibition of PLD activity has been shown to attenuate MTI as well as ETI. In this study, we systematically tested single and double knockouts in all 12 genes encoding PLDs in Arabidopsis thaliana for effects on ETI and MTI. No single PLD could be linked to ETI triggered by recognition of effectors secreted by the bacterium Pseudomonas syringae. However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition. In addition some pld mutants were more sensitive to n-butanol than wild type. Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR. Only knockout of PLDδ caused a loss of MTI-induced cell wall based defense against the non-host powdery mildew Erysiphe pisi. This is thus in stark contrast to the involvement of a multitude of PLD isoforms in the HR triggered by AvrRpm1 recognition.