Search

The carbonate chemistry of sea ice is known to play a role in global carbon cycles, but its importance is uncertain in part due to disparities in reported results. Variability in physical and biological drivers is usually invoked to explain differences between studies. In the Canadian Arctic Archipelago, “invisible polynyas” – areas of strong currents, thin ice, and potentially high biological productivity – are examples of extreme spatial variability. We used an invisible polynya as a natural laboratory to study the effects of inferred initial ice formation conditions, ice growth rate, and algal biomass on the distribution of carbonate species by collecting enough cores to perform a statistical comparison between sites located within, and just outside of, a polynya near Iqaluktuttiaq (Cambridge Bay, Nunavut, Canada). At both sites, the uppermost 10 cm ice horizon showed evidence of CO2 off-gassing, while carbonate distributions in the middle and bottommost 10 cm horizons largely followed the salinity distribution. In the polynya, the upper ice horizon had significantly higher bulk total inorganic carbon (TIC), total alkalinity (TA), and salinity potentially due to freeze-up conditions that favoured frazil ice production. The middle ice horizons were statistically indistinguishable between sites, suggesting that ice growth rate is not an important factor for the carbonate distribution under mid-winter conditions. The thicker (non-polynya) site experienced higher algal biomass, TIC, and TA in the bottom horizon. Carbonate chemistry in the bottom horizon could largely be explained by the salinity distribution, with the strong currents at the polynya site potentially playing a role in desalinization; biology appeared to exert only a minor control, with some evidence that the ice algae community was net heterotrophic. We did see evidence of calcium carbonate precipitation but with little impact on the TIC:TA ratio and little difference between sites. Because differences were constrained to relatively thin layers at the top and bottom, vertically averaged values of TIC, TA, and especially the TIC:TA ratio were not meaningfully different between sites. This provides some justification for using a single bulk value for each parameter when modelling sea ice effects on ocean chemistry at coarse resolution. Exactly what value to use (particularly for the TIC:TA ratio) likely varies by region but could potentially be approximated from knowledge of the source seawater and sea ice salinity. Further insights await a rigorous intercomparison of existing data.

International audience; One of the most striking ecological divides on Earth is between marine and nearby freshwater environments, as relatively few taxa can move between the two. Microbial eukaryotes contribute to biogeochemical and energy cycling in both fresh and marine waters, with little species overlap between the two ecosystems. Arctic and sub-Arctic marine systems are relatively fresh compared to tropical and temperate systems, but details of microbial eukaryote communities along river-to-sea transitions are poorly known. To bridge this knowledge gap, we investigated three river-to-sea transitions (Nelson, Churchill, and Great Whale Rivers) in sub-Arctic Hudson Bay through 18S rRNA amplicon sequencing to identify microbial eukaryotes along the salinity and biogeochemical gradients. Salinity acted as the principal dispersal barrier preventing freshwater microorganisms from colonizing marine coastal waters, with microbial eukaryote communities of the three rivers clustering together. Just offshore, communities clustered by coastal regions associated with nutrient concentrations. Analysis of indicator species revealed that communities in the nitrate-depleted coastal water off the Churchill and Great Whale Rivers were dominated by heterotrophic taxa and small photosynthetic protists. In contrast, the Nelson offshore community was characterized by a high proportion of the diatom Rhizosolenia. A distinct community of heterotrophic protists was identified in the three estuarine transition zones, suggesting specialized estuarine communities. Such specialization was most marked in the Nelson River system that was sampled more intensely and showed estuarine circulation.The autochthonous community was composed of the bacterial grazers Katablepharis, Mataza, and Cryothecomonas, as well as brackish species of the diatoms Skeletonema and Thalassiosira. These findings suggest that flow regulation on the Nelson River that modifies estuarine circulation would affect estuarine community composition and distribution in the transition zone.

A new species of monstrilloid copepod, Monstrillopsis planifrons sp. n., is described from an adult female that was collected beneath snow-covered sea ice during the 2014 Ice Covered Ecosystem – CAMbridge bay Process Study (ICE-CAMPS) in Dease Strait of the Canadian Arctic Archipelago. Currently, up to six species of this order are known to occur in polar latitudes. The new species described herein shares similarities with Monstrillopsis dubia (Scott, 1904) but differs in its body proportions and cephalothorax ornamentation; the cephalothorax is covered by minute scattered papillae on dorsal and ventral surfaces; this species has a reduced fifth leg endopod, fifth leg exopod armed with three setae, antennule with fused segments 3–4, and the genital double-somite bears unique posterolateral processes. This is the second species of this genus recorded in the Arctic, after Monstrillopsis ferrarii (Suárez-Morales & Ivanenko, 2004), described from the White Sea, and is the first record of Monstrillopsis in Canadian waters. With the addition of this new species and the recognition of Monstrillopsis bernardensis comb. nov. as a member of this genus, the number of nominal species is now 15. Overall, this genus has a tendency to be distributed in temperate and cold waters, while only three species have been found in tropical and subtropical latitudes.

The abundance of diatoms and heterotrophic bacteria in sea ice rapidly increases during the spring. However, the number and activity of these microorganisms vary with changing environmental conditions and potentially the taxonomic composition of the algal community during this time. In this study, we assessed the spring bottom-ice community composition in Dease Strait, Nunavut, and investigated potential controls of chlorophyll a (chl a), particulate organic carbon (POC), cell abundance, and production from early March until early June. We found that using flow cytometry to estimate photosynthetic nanoeukaryote (2–20 μm) abundance gave results very similar to light microscopy counts, except when pennate diatoms with lengths close to 20 μm, the maximum size detected by flow cytometry, were abundant. Using the average abundance of nanoeukaryotes from the two methods, we documented a change in the size of cells comprising the ice algal community over the spring, from largely pico- (

A number of common sterols were identified in sea ice diatoms from the Arctic and the Antarctic. The main sterols in floating sea ice algal aggregates collected from Resolute Passage (Canadian Arctic) and Nansen Basin (North Svalbard) in 2012 were 22E-dehydrocholesterol, cholesterol, epi-brassicasterol, 24-methylenecholesterol and 24-ethylcholesterol, although the distribution varied between the two locations, likely reflecting compositional differences in diatom taxa. The three major sterols in cells of Berkeleya adeliensis picked from a melted sea ice core collected from Ryder Bay in the Antarctic Peninsula in 2014, were 24-ethylcholesterol, cholesterol and 22E-dehydrocholesterol. We suggest that certain sea ice diatoms may thus contribute to the sedimentary budget of common sterols in seasonally sea ice-covered locations following ice melt.

Sea ice algae are an important contributor of primary production in the Arctic ecosystem. Within the bottom-ice environment, access to nutrients from the underlying ocean is a major factor controlling production, phenology, and taxonomic composition of ice algae. Previous studies have demonstrated that tides and currents play an important role in driving the flux of nutrients to bottom-ice algal communities when biological demand during the spring bloom is high. In this study we investigate how surface currents under landfast first-year ice influence nutrient supply based on stoichiometric composition, algal chlorophyll a biomass and species composition during spring 2016, in Dease Strait, Nunavut. Stronger water dynamics over a shoaled and constricted strait dominated by tidal currents (tidal strait) supported turbulent flow more than 85% of the deployment duration in comparison to outside the tidal strait in an embayment where turbulent flow was only evidenced a small percentage (

Light is a major environmental cue affecting various physiological and metabolic processes in plants. Although plant photoreceptors are well characterized, the mechanisms by which light regulates downstream responses are less clear. In Arabidopsis thaliana, the accumulation of photoprotective anthocyanin pigments is light dependent, and the R2R3 MYB transcription factor MYB75/PAP1 regulates anthocyanin accumulation. Here, we report that MYB75 interacts with and is phosphorylated by MAP KINASE4 (MPK4). Their interaction is dependent on MPK4 kinase activity and is required for full function of MYB75. MPK4 can be activated in response to light and is involved in the light-induced accumulation of anthocyanins. We show that MPK4 phosphorylation of MYB75 increases its stability and is essential for light-induced anthocyanin accumulation. Our findings reveal an important role for a MAPK pathway in light signal transduction.

Genetics studies the structure/function of genes via the characterization of their mutant phenotypes. In plants, a readily scorable mutant phenotype comprises macroscopic lesions symptomatic of disease in the absence of pathogens. Such mutants therefore exhibit autoimmune phenotypes. Many of these mutants are considered to be associated with immunity and the corresponding genes have been described as 'negative regulators' of immunity and/or cell death. Pathogens deliver effectors into host cells to increase infectivity by modifying or removing host proteins. Plants detect effectors via nucleotide-binding, leucine-rich repeat (NLR) immune receptors, which monitor host effector targets. In response to effector-mediated target tampering, NLR proteins potentiate immunity. The guard hypothesis proposes that NLRs 'guard' host 'guardees' targeted by pathogen effectors. An obvious corollary to this guard model is that forms of plant autoimmunity are a result of inappropriate NLR protein activation. In this review, we discuss what is known about some of the 'negative regulators' of immunity, and propose simple strategies that may help to characterize autoimmune mutants.

The importance of newly formed sea ice in spring is likely to increase with formation of leads in a more dynamic Arctic icescape. We followed the ice algal species succession in young ice (≤ 0.27 m) in spring at high temporal resolution (sampling every second day for 1 month in May–June 2015) in the Arctic Ocean north of Svalbard. We document the early development of the ice algal community based on species abundance and chemotaxonomic marker pigments, and relate the young-ice algal community to the communities in the under-ice water column and the surrounding older ice. The seeding source seemed to vary between algal groups. Dinoflagellates were concluded to originate from the water column and diatoms from the surrounding older ice, which emphasizes the importance of older ice as a seeding source over deep oceanic regions and in early spring when algal abundance in the water column is low. In total, 120 taxa (80 identified to species or genus level) were recorded in the young ice. The protist community developed over the study period from a ciliate, flagellate, and dinoflagellate dominated community to one dominated by pennate diatoms. Environmental variables such as light were not a strong driver for the community composition, based on statistical analysis and comparison to the surrounding thicker ice with low light transmission. The photoprotective carotenoids to Chl a ratio increased over time to levels found in other high-light habitats, which shows that the algae were able to acclimate to the light levels of the thin ice. The development into a pennate diatom-dominated community, similar to the older ice, suggests that successional patterns tend toward ice-associated algae fairly independent of environmental conditions like light availability, season or ice type, and that biological traits, including morphological and physiological specialization to the sea ice habitat, play an important role in colonization of the sea ice environment. However, recruitment of ice-associated algae could be negatively affected by the ongoing loss of older ice, which acts as a seeding repository. Copyright © 2018 Kauko, Olsen, Duarte, Peeken, Granskog, Johnsen, Fernández-Méndez, Pavlov, Mundy and Assmy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

During the N-ICE2015 drift expedition north-west of Svalbard, we observed the establishment and development of algal communities in first-year ice (FYI) ridges and at the snow-ice interface. Despite some indications of being hot spots for biological activity, ridges are under-studied largely because they are complex structures that are difficult to sample. Snow infiltration communities can grow at the snow-ice interface when flooded. They have been commonly observed in the Antarctic, but rarely in the Arctic, where flooding is less common mainly due to a lower snow-to-ice thickness ratio. Combining biomass measurements and algal community analysis with under-ice irradiance and current measurements as well as light modeling, we comprehensively describe these two algal habitats in an Arctic pack ice environment. High biomass accumulation in ridges was facilitated by complex surfaces for algal deposition and attachment, increased light availability, and protection against strong under-ice currents. Notably, specific locations within the ridges were found to host distinct ice algal communities. The pennate diatoms Nitzschia frigida and Navicula species dominated the underside and inclined walls of submerged ice blocks, while the centric diatom Shionodiscus bioculatus dominated the top surfaces of the submerged ice blocks. Higher light levels than those in and below the sea ice, low mesozooplankton grazing, and physical concentration likely contributed to the high algal biomass at the snow-ice interface. These snow infiltration communities were dominated by Phaeocystis pouchetii and chain-forming pelagic diatoms (Fragilariopsis oceanica and Chaetoceros gelidus). Ridges are likely to form more frequently in a thinner and more dynamic ice pack, while the predicted increase in Arctic precipitation in some regions in combination with the thinning Arctic icescape might lead to larger areas of sea ice with negative freeboard and subsequent flooding during the melt season. Therefore, these two habitats are likely to become increasingly important in the new Arctic with implications for carbon export and transfer in the ice-associated ecosystem. Copyright © 2018 Fernández-Méndez, Olsen, Kauko, Meyer, Rösel, Merkouriadi, Mundy, Ehn, Johansson, Wagner, Ervik, Sorrell, Duarte, Wold, Hop and Assmy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)

DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity. However, other immune responses may suppress DNA damage to maintain host genome integrity. Here, we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding, Leucine-rich-repeat immune Receptors (NLRs). SNI1 (suppressor of npr1-1, inducible 1), a subunit of the structural maintenance of chromosome (SMC) 5/6 complex, was reported to be a negative regulator of systemic acquired resistance (SAR) and to be necessary for controlling DNA damage. We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1. Similar to sni1, elevated DNA damage is seen in other autoimmune mutants with cell death lesions, including camta3, pub13 and vad1, but not in dnd1, an autoimmune mutant with no visible cell death. We find that as in sni1, DNA damage in camta3 is EDS1-dependent, but that it is also NLR-dependent. Using the NLR RPM1 as a model, we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors. We also find that the expression of DNA damage repair (DDR) genes in mutants with cell death lesions is down regulated, suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair. Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation., Author summary DNA is constantly subjected to damaging agents that can cause mutations, disease and cell death. Not surprisingly, cells have evolved mechanisms to repair damaged DNA and preserve its integrity. Recent reports suggested that the innate immune system in plants might actively damage host DNA as a mechanism to boost resistance to pathogens. This process was shown to be mediated by the phytohormone Salicylic Acid and the DNA damage repair protein and immunity regulator SNI1. In contrast, we report that the DNA damage observed in sni1 is independent of its role in DNA damage repair and is instead linked to autoimmunity and shared with other, unrelated autoimmune mutants. We demonstrate, genetically, that cellular dismantling triggered by NLR-mediated immunity is a more plausible explanation for the DNA damage observed in those mutants. Our results provide clarification and new insight into the role of DNA damage and immunity.

This study reports on the temporal variations in algal and bacterial metabolism of dissolved dimethylsulfoniopropionate (DMSPd) in Arctic ice-covered waters in response to the release of organic matter (OM) from the sea ice and the onset of under-ice phytoplankton growth. Sampling took place between 21 May and 21 June 2012 at a station located in Resolute Passage. A snow and ice melt event was accompanied by an important release of OM and total DMSP from the bottom ice to the water column. This input of OM coincided with increases in DMSPd and DMSPd loss rate constant at the ice-water interface and, 2 days later, with increases in DMSPd and bacter- ial dimethylsulfide (DMS) yields from DMSPd at 0.5 m under the ice. The different microbial re - sponses suggest that DMSPd-rich brines were released first, followed by the release of sympagic algae due to ice melt. In both cases, the changes in DMSPd metabolism resulted in an increase in gross DMS production from 0.15 to 1.9 nmol l �1 d �1 . The initiation of phytoplankton growth resulted in increases in bacterial abundance, DMSPd loss-rate constant and DMSP-sulfur assimilation. In contrast, DMS yield remained low during the onset of phytoplankton growth, indicating that bac- teria used DMSP as a carbon and sulfur source. These results show that ice DMSPd can be rapidly (

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

We measured the concentrations of selected lipids and some of their degradation products in a time series of sea ice samples collected from Resolute Bay in the Canadian Arctic in 2012. The identification of specific tracers of photo- and autoxidation reactions provided evidence of both abiotic processes acting on organic matter, although none of the lipids appeared sensitive to significant biodegradation. Some differences in lipid reactivity were observed between the upper (3–10 cm) and lower (0–3 cm) sections of the cores, possibly as a result of increased exchange at the ice–water boundary for the latter. In terms of photodegradation, the phytyl side chain of chlorophyll and a tri-unsaturated highly branched isoprenoid (HBI) alkene were most affected, 24-methylenecholesterol (from diatoms) was more susceptible to photodegradation than brassicasterol (from diatoms and prymnesiophytes), while sitosterol and cholesterol, likely from a range of sources, were largely unaffected. With respect to autoxidation, the reactivity trend for the sterols was reversed, with significant autoxidation of sitosterol and cholesterol, but not of 24-methylenecholesterol or brassicasterol. The phytyl side chain of chlorophyll and a highly branched isoprenoid triene were also particularly susceptible to autoxidation. In contrast, the diatom fatty acid, C16:1, was not substantially altered by any degradation pathway. By measuring temporal changes in the proportions of specific tracers of each degradation process, we provide evidence that such abiotic reactions take place via sequential photooxidation and autoxidation, with homolytic cleavage of photochemically produced hydroperoxides leading to autoxidation of initial substrates and subsequent oxidation products. The observation of significant abiotic degradation of several lipids appears to take place despite the low temperature in sea ice (ca. 0 °C) and should be considered carefully alongside other removal processes involving organic matter in marine systems.

We have used thermal source Fourier Transform Infrared (FTIR) microtomographic imaging to compare sea ice diatoms growing under different light conditions. A prototype tomography accessory was designed to have sufficient degrees of freedom to align any tilted cylindrical sample relative to the axis of rotation, minimizing the off-axis path traced during rotation. The lightweight device rests on the motorized stage to position the sample in the field-of-view and enable mosaic imaging. Reconstruction routines were tested with simulated and real phantoms, to assess limitations in the Radon back-projection method employed. The distribution and abundance of biochemicals is analysed for targets larger than a single FPA tile. Two and three dimensional (2D and 3D) FTIR spectrochemical images were obtained with a Focal Plane Array (FPA, nominal 1.1 μm pixel edges) for phantoms (polystyrene beads in polyvinyl alcohol matrix) and diatom cells harvested from land fast, first-year ice sites in Resolute Passage (74 43.628'N; 95 33.330'W) and Dease Strait (69° 1.11'N; 105° 21.29'W), Nunavut, Canada. The analysis of relative concentrations of organic matter within the encapsulating silica frustules of diatoms is important for a better understanding of both the physiological state and the individual cellular response to environmental pressures. Analysis of 3D FTIR images of Nitzschia frigida collected from beneath high (17-19 cm) and low (3-7 cm) snow depth revealed higher concentrations of lipids in diatoms collected under low snow cover, uniquely based on spectroscopically determined total 3D cell volume and biochemical content.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

A MAP kinase pathway below a chitin receptor in the moss Physcomitrella patens induces immune responses including rapid growth inhibition, a novel fluorescence burst, and cell wall depositions. The molecular mechanisms producing these three responses are currently unknown but warrant further investigation in this simple model system. Here we describe qualitative, time-lapse, and quantitative assays to monitor and measure these responses.

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts.

Summary To establish infection, pathogens deploy effectors to modify or remove host proteins. Plant immune receptors with nucleotide-binding, leucine-rich repeat domains (NLRs) detect these modifications and trigger immunity. Plant NLRs thus guard host "guardees." A corollary is that autoimmunity may result from inappropriate NLR activation because mutations in plant guardees could trigger corresponding NLR guards. To explore these hypotheses, we expressed 108 dominant-negative (DN) Arabidopsis NLRs in various lesion mimic mutants, including camta3 , which exhibits autoimmunity. CAMTA3 was previously described as a negative regulator of immunity, and we find that autoimmunity in camta3 is fully suppressed by expressing DNs of two NLRs, DSC1 and DSC2. Additionally, expression of either NLR triggers cell death that can be suppressed by CAMTA3 expression. These findings support a model in which DSC1 and DSC2 guard CAMTA3, and they suggest that other negative regulators of immunity may similarly represent guardees.

This study presents temporal variations in concentrations of chlorophyll a (Chl a), particulate and dissolved dimethylsulfoniopropionate (DMSPp and DMSPd) in the sea ice and underlying water column in the Canadian Arctic Archipelago during the spring of 2010 and 2011. During both years, bottom ice Chl a, DMSPp and DMSPd concentrations were high (up to 1328 µg L−1, 15,082 nmol L−1, and 6110 nmol L−1, respectively) in May and decreased thereafter. The release of bottom ice algae and DMSPp in the water column was gradual in 2010 and rapid (8 days) in 2011. Bottom brine drainage during the presnowmelt period in 2010 and a rapid loss of the snow cover in 2011 coinciding with rain events explain most of the difference between the 2 years. During both years, less than 13% of the DMSPd lost from the ice was detected in the water column, suggesting a rapid microbial consumption. An under-ice diatom bloom developed in both years. In 2010, the bloom was dominated by centric diatoms while in 2011 pennates dominated, likely reflecting seeding by ice algae following the faster snowmelt progression induced by rainfall events in 2011. Both under-ice blooms were associated with high DMSPp concentrations (up to 185 nmol L−1), but pennate diatoms showed DMSPp/Chl a ratios twice higher than centrics. These results highlight the key role of snowmelt and precipitation on the temporal pattern of ice-DMSP release to the water column and on the timing, taxonomic composition, and DMSP content of phytoplankton under-ice blooms in the Arctic.

Summary The accelerated cell death 11 ( acd11 ) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1-phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, "sandwich" topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily.

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

Sea ice algae are a characteristic feature in ice-covered seas, contributing a significant fraction of the total primary production in many areas and providing a concentrated food source of high nutritional value to grazers in the spring. Algae respond to physical changes in the sea ice environment by modifying their cellular carbon, nitrogen and pigment content, and by adjusting their photophysiological characteristics. In this study we examined how the ratios of particulate organic carbon (POC) to nitrogen (PON), and POC to chlorophyll a (chl a), responded to the evolving snow-covered sea ice environment near Cambridge Bay, Nunavut, during spring 2014. We also estimated photosynthesis-irradiance (PI) curves using oxygen-optodes and evaluated the resulting time-series of PI parameters under thin and thick snow-covered sites. There were no significant differences in PI parameters between samples from different overlying snow depths, and only the maximum photosynthetic rates in the absence of photoinhibition (PsB) and photoacclimation (IS) parameters changed significantly over the spring bloom. Furthermore, we found that both these parameters increased over time in response to increasing percent transmission of photosynthetically active radiation (TPAR) through the ice, indicating that light was a limiting factor of photosynthesis and was an important driver of temporal (over the spring) rather than spatial (between snow depths) variability in photophysiological response. However, we note that spatial variability in primary production was evident. Higher TPAR over the spring and under thin snow affected the composition of algae over both time and space, causing greater POC:chl a estimates in late spring and under thin snow cover. Nitrogen limitation was pronounced in this study, likely reducing PsB and algal photosynthetic rates, and increasing POC:PON ratios to over six times the Redfield average. Our results highlight the influence of both light and nutrients on ice algal biomass composition and photophysiology, and suggest a limitation by both resources over a diel period.

The lipid content of sea ice samples collected in 2011 and 2012 from Resolute Passage in the Canadian Arctic Archipelago was measured and compared with related samples obtained from the Amundsen Gulf in 2008. The highly branched isoprenoid (HBI) sea ice biomarker, IP 25 , was found in sea ice samples from each study, consistent with its formation by diatoms during the spring bloom. Our analysis also revealed the occurrence of a number of sterols in Arctic sea ice and these were rigorously identified and quantified for the first time. Concentrations of IP 25 and sterols exhibited some variability between sampling studies, with somewhat higher values in samples from Resolute in 2012 than for the other two datasets, consistent with a general increase in biomass; however, major differences in biomarker concentration between sampling studies were not observed. An estimate of the proportion of Arctic sea ice diatoms that produce IP 25 (ca. 1–5%) was obtained by comparison of the concentration of IP 25 in the samples with those in laboratory cultures of known HBI-producing diatoms and cell enumeration in selected sea ice samples. The estimate is similar to the proportion of Haslea spp. in the same samples, providing further support to the suggestion that at least some species of the Haslea genus may be responsible for the biosynthesis of IP 25 and related HBI diatom lipids in Arctic sea ice and that IP 25 is made by a relatively small proportion of sea ice diatoms. In contrast, median sterol/IP 25 values were all substantially higher than those in cultures of HBI-producing diatoms, suggesting that sterols are made by the majority of sea ice diatoms. The sterol/IP 25 ratio was quite variable between locations and samples, likely as a result of differences in diatom assemblage; however, a comparison of individual and median sterol/IP 25 values in sea ice with those from surface sediments from different Arctic regions demonstrated that sterols from sea ice diatoms may, in some cases, have a significant impact on the sedimentary budget. This should be considered carefully for quantitative estimates of palaeo sea ice reconstruction using methods such as the PIP 25 index, which are based on the relative concentrations of IP 25 and sterols in Arctic marine sediment cores.

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences. Co-expression analysis across publicly available microarray experiments identified 25 genes co-expressed with ATAF1. The promoter regions of ATAF1 co-expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis., Highlights • Consensus DNA-binding sites of the Arabidopsis transcription factor ATAF1 were identified. • The ATAF1 co-expression cluster is enriched for ATAF1 consensus binding sites • ABA biosynthetic gene NCED3 is co-expressed with ATAF1. • Plants over-expressing ATAF1 have increased ABA hormone levels. • ATAF1 binds in vivo to the promoter of NCED3 to activate expression.

International audience; The abiotic degradation state of sea ice algae released during a late spring ice melt process was determined by sampling the underlying waters and measuring certain well-known algal lipids and their oxidation products, including those derived from epi-brassicasterol, 24-methylenecholesterol, palmitoleic acid and the phytyl side-chain of chlorophyll. More specifically, parent lipids and some of their oxidation products were quantified in suspended (collected by filtration) and sinking (collected with sediment traps at 5 and 30 m) particles from Resolute Passage (Canada) during a period of spring ice melt in 2012 and the outcomes compared with those obtained from related sea ice samples analyzed previously. Our data show that suspended cells in the near surface waters appeared to be only very weakly affected by photooxidative processes, likely indicative of a community of unaggregated living cells with high seeding potential for further growth. In contrast, we attribute the strong photooxidation state of the organic matter in the sediment traps deployed at 5 m to the presence of senescent and somewhat aggregated sea ice algae that descended only relatively slowly within the euphotic zone, and was thus susceptible to photochemical degradation. On the other hand, the increased abiotic preservation of the sinking material collected in the sediment traps deployed at 30 m, likely reflected more highly aggregated senescent sea ice algae that settled sufficiently rapidly out of the euphotic zone to avoid significant photooxidation. This better-preserved sinking material in the deeper sediment traps may therefore contribute more strongly to the underlying sediments. A three-component conceptual scheme summarizing the abiotic behavior of Arctic sea ice algae in underlying waters is proposed

During summer 2008, as part of the Circumpolar Flaw Lead system study, we measured phytoplankton photosynthetic parameters to understand regional patterns in primary productivity, including the degree and timescale of photoacclimation and how variability in environmental conditions influences this response. Photosynthesis–irradiance measurements were taken at 15 sites primarily from the depth of the subsurface chlorophyll a (Chl a) maximum (SCM) within the Beaufort Sea flaw lead polynya. The physiological response of phytoplankton to a range of light levels was used to assess maximum rates of carbon (C) fixation (P m * ), photosynthetic efficiency (α *), photoacclimation (E k), and photoinhibition (β *). SCM samples taken along a transect from under ice into open water exhibited a >3-fold increase in α * and P m * , showing these parameters can vary substantially over relatively small spatial scales, primarily in response to changes in the ambient light field. Algae were able to maintain relatively high rates of C fixation despite low light at the SCM, particularly in the large (>5 μm) size fraction at open water sites. This may substantially impact biogenic C drawdown if species composition shifts in response to future climate change. Our results suggest that phytoplankton in this region are well acclimated to existing environmental conditions, including sea ice cover, low light, and nutrient pulses. Furthermore, this photoacclimatory response can be rapid and keep pace with a developing SCM, as phytoplankton maintain photosynthetic rates and efficiencies in a narrow “shade-acclimated” range.

Biological characteristics of ice-associated algal communities were studied in Darnley Bay (western Canadian Arctic) during a 2-week period in July 2008 when the landfast ice cover had reached an advanced stage of melt. We found two distinct and separate algal communities: (1) an interior ice community confined to brine channel networks beneath white ice covers; and (2) an ice melt water community in the brackish waters of both surface melt ponds and the layer immediately below the ice cover. Both communities reached maximum chlorophyll a concentrations of about 2.5 mg m−3, but with diatoms dominating the interior ice while flagellates dominated the melt water community. The microflora of each community was diverse, containing both unique and shared algal species, the latter suggesting an initial seeding of the ice melt water by the bottom ice community. Absorption characteristics of the algae indicated the presence of mycosporine-like amino acids (MAAs) and carotenoid pigments as a photoprotective strategy against being confined to high-light near-surface layers. Although likely not contributing substantially to total annual primary production, these ice-associated communities may play an important ecological role in the Arctic marine ecosystem, supplying an accessible and stable food source to higher trophic levels during the period of ice melt.

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Early summer in the Arctic with extensive ice melt and break-up represents a dramatic change for sympagic–pelagic fauna below seasonal sea ice. As part of the International Polar Year-Circumpolar Flaw Lead system study (IPY-CFL), this investigation quantified zooplankton in the meltwater layer below landfast ice and remaining ice fauna below melting ice during June (2008) in Franklin Bay and Darnley Bay, Amundsen Gulf, Canada. The ice was in a state of advanced melt, with fully developed melt ponds. Intense melting resulted in a 0.3- to 0.5-m-thick meltwater layer below the ice, with a strong halocline to the Arctic water below. Zooplankton under the ice, in and below the meltwater layer, was sampled by SCUBA divers. Dense concentrations (max. 1,400 ind. m−3) of Calanus glacialis were associated with the meltwater layer, with dominant copepodid stages CIV and CV and high abundance of nauplii. Less abundant species included Pseudocalanus spp., Oithona similis and C. hyperboreus. The copepods were likely feeding on phytoplankton (0.5–2.3 mg Chl-a m−3) in the meltwater layer. Ice amphipods were present at low abundance (

Variations in the concentrations of the sea ice diatom biomarker, IP25 (Ice Proxy with 25 carbon atoms), were measured in the bottom 10 cm of sea ice collected from the eastern Beaufort Sea and Amundsen Gulf from January to June 2008, as part of the International Polar Year–Circumpolar Flaw Lead system study. Temporal and vertical changes in IP25 concentrations were compared against other biomarkers and indicators of ice algal production. IP25 was not detected in sea ice samples collected from mid-winter to early spring, likely as a result of light-limiting conditions for algal growth and accumulation. From early March to mid-June, IP25 concentrations correlated well with those of fatty acids (r = 0.79; P 5% which is consistent with the hypothesis that brine channel connectivity limits the internal colonisation of sea ice by diatoms. Maximum IP25 concentrations occurred at 1–3 cm from the ice–water interface providing further evidence for a selective sea ice diatom origin for this biomarker. In contrast, vertical concentration profiles for fatty acids and sterols indicated mixed sources for these biomarkers.

SummaryAutophagy has been implicated as a prosurvival mechanism to restrict programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. This model is based on the observation that HR lesions spread in plants with reduced autophagy gene expression. Here, we examined receptor-mediated HR PCD responses in autophagy-deficient Arabidopsis knockout mutants (atg), and show that infection-induced lesions are contained in atg mutants. We also provide evidence that HR cell death initiated via Toll/Interleukin-1 (TIR)-type immune receptors through the defense regulator EDS1 is suppressed in atg mutants. Furthermore, we demonstrate that PCD triggered by coiled-coil (CC)-type immune receptors via NDR1 is either autophagy-independent or engages autophagic components with cathepsins and other unidentified cell death mediators. Thus, autophagic cell death contributes to HR PCD and can function in parallel with other prodeath pathways.

The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death, and transgenic plants showed normal induction of hypersensitive cell death upon infection with avirulent strains of Pseudomonas syringae. The significance of these findings with respect to the function(s) of ACD11 in sphingolipid transport and cell death regulation is discussed.

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

The Arabidopsis (Arabidopsis thaliana) MKK1 and MKK2 mitogen-activated protein kinase kinases have been implicated in biotic and abiotic stress responses as part of a signaling cascade including MEKK1 and MPK4. Here, the double loss-of-function mutant (mkk1/2) of MKK1 and MKK2 is shown to have marked phenotypes in development and disease resistance similar to those of the single mekk1 and mpk4 mutants. Because mkk1 or mkk2 single mutants appear wild type, basal levels of MPK4 activity are not impaired in them, and MKK1 and MKK2 are in part functionally redundant in unchallenged plants. These findings are confirmed and extended by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both redundant and unique functions for MKK1 and MKK2.

Programmed cell death (PCD) occurs during vegetative and reproductive plant growth, as typified by autumnal leaf senescence and the terminal differentiation of the endosperm of cereals which provide our major source of food. PCD also occurs in response to environmental stress and pathogen attack, and these inducible PCD forms are intensively studied due their experimental tractability. In general, evidence exists for plant cell death pathways which have similarities to the apoptotic, autophagic and necrotic forms described in yeast and metazoans. Recent research aiming to understand these pathways and their molecular components in plants are reviewed here.

Microscale photographs were taken of the ice bottom to examine linkages of algal chlorophyll a (chl a) biomass distribution with bottom ice features in thick Arctic first-year sea ice during a spring field program which took place from May 5 to 21, 2003. The photographic technique developed in this paper has resulted in the first in situ observations of microscale variability in bottom ice algae distribution in Arctic first-year sea ice in relation to ice morphology. Observations of brine channel diameter (1.65–2.68 mm) and number density (5.33–10.35 per 100 cm2) showed that the number of these channels at the bottom of thick first-year sea ice may be greater than previously measured on extracted ice samples. A variogram analysis showed that over areas of low chl a biomass (≤20.7 mg chl a m−2), patchiness in bottom ice chl a biomass was at the scale of brine layer spacing and small brine channels (∼1–3 mm). Over areas of high chl a biomass (≥34.6 mg chl a m−2), patchiness in biomass was related to the spacing of larger brine channels on the ice bottom (∼10–26 mm). Brine layers and channels are thought to provide microscale maxima of light, nutrient replenishment and space availability which would explain the small scale patchiness over areas of low algal biomass. However, ice melt and erosion near brine channels may play a more important role in areas with high algal biomass and low snow cover.

MAP kinase (MPK) cascades in Arabidopsis thaliana and other vascular plants are activated by developmental cues, abiotic stress, and pathogen infection. Much less is known of MPK functions in nonvascular land plants such as the moss Physcomitrella patens. Here, we provide evidence for a signaling pathway in P. patens required for immunity triggered by pathogen associated molecular patterns (PAMPs). This pathway induces rapid growth inhibition, a novel fluorescence burst, cell wall depositions, and accumulation of defense-related transcripts. Two P. patens MPKs (MPK4a and MPK4b) are phosphorylated and activated in response to PAMPs. This activation in response to the fungal PAMP chitin requires a chitin receptor and one or more MAP kinase kinase kinases and MAP kinase kinases. Knockout lines of MPK4a appear wild type but have increased susceptibility to the pathogenic fungi Botrytis cinerea and Alternaria brassisicola. Both PAMPs and osmotic stress activate some of the same MPKs in Arabidopsis. In contrast, abscisic acid treatment or osmotic stress of P. patens does not activate MPK4a or any other MPK, but activates at least one SnRK2 kinase. Signaling via MPK4a may therefore be specific to immunity, and the moss relies on other pathways to respond to osmotic stress.

The Arabidopsis accelerated-cell-death11 (acd11) mutant provides a genetic model for studying localized cell suicide to halt the spread of pathogen infection in plants. Recently, we showed that ACD...

Multi‐layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen‐associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de‐repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses.

*Summary Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA–ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4–EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/ mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, and their components are encoded by highly conserved genes. The recent availability of genome sequences for rice and poplar now makes it possible to examine how well the previously described Arabidopsis MAPK and MAPKK gene family structures represent the broader evolutionary situation in plants, and analysis of gene expression data for MPK and MKK genes in all three species allows further refinement of those families, based on functionality. The Arabidopsis MAPK nomenclature appears sufficiently robust to allow it to be usefully extended to other well-characterized plant systems.

We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H+-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

mRNA of the putative Arabidopsis flavin mono-oxygenase encoded by At1g19250 (here designated AtFMO GenBank accession no. NM_101783) accumulated to high levels in the acd11 and lsd1 mutants that exhibit run-away cell death, but not in mutants with constitutive defense responses. AtFMO mRNA accumulation occurred at the transcriptional level because a fusion between the AtFMO promoter and the GUS reporter was induced in acd11 compared to wild type. AtFMO::GUS activity also appeared significantly earlier in wild type plants inoculated with avirulent strains of Pseudomonas syringae compared to a virulent strain. In addition, a close barley homolog was identified by differential display and shown also be induced by pathogen infection. AtFMO::GUS was induced by the fungal toxin fumonisin B1 and by superoxide generation, but not by treatments with hydrogen peroxide, ozone or nitric oxide. During normal development, AtFMO::GUS activity increased from very low, basal levels in young leaves to fairly high levels in the senescing regions of old leaves. The correlation between AtFMO expression and occurrence of cell death suggests that AtFMO mRNA and the AtFMO::GUS reporter are amenable markers for certain forms of defense and cell death. # 2005 Elsevier Ireland Ltd. All rights reserved.

The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discussed. (Managing editor: Ping He)

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.

The Arabidopsis MYB68 gene encodes a MYB family protein with N-terminal R2R3 DNA-binding domains. Analyses of MYB68 expression by RNA blot and a transposant gene-trap MYB68::GUS reporter indicated that MYB68 is expressed specifically in root pericycle cells. Root cultures of the myb68 mutant, caused by the gene trap insertion in the first MYB68 exon, produced increased biomass and lignin levels compared to wild type. Under high temperature regimes, MYB68::GUS activity was elevated in roots, while vegetative growth of myb68 mutants was reduced compared to wild type. These data suggest that MYB68 is involved in some step(s) in root development. The closest MYB68 homolog, MYB84, exhibited an overlapping expression pattern in pericycle cells, suggesting that their functions may be partly redundant.

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.