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bioRxiv2018

Transcriptional landscape of soybean (Glycine max) embryonic axes during germination in the presence of paclobutrazol, a gibberellin biosynthesis inhibitor

Gibberellins (GA) are key positive regulators of seed germination. Although the GA effects on seed germination have been studied in a number of species, little is known about the transcriptional reprogramming modulated by GA during this phase in species other than Arabidopsis thaliana. Here we report the transcriptome analysis of soybean embryonic axes during germination in the presence of paclobutrazol (PBZ), a GA biosynthesis inhibitor. We found a number of differentially expressed cell wall metabolism genes, supporting their roles in cell expansion during germination. Several genes involved in the biosynthesis and signaling of other phytohormones were also modulated, indicating an intensive hormonal crosstalk at the embryonic axis. We have also found 26 photosynthesis genes that are up-regulated by PBZ at 24 hours of imbibition (HAI) and down-regulated at 36 HAI, which led us to suggest that this is part of a strategy to implement an autotrophic growth program in the absence of GA-driven mobilization of reserves. Finally, 30 transcription factors (mostly from the MYB, bHLH and bZIP families) that are down-regulated by PBZ and are likely downstream GA targets that will drive transcriptional changes during germination.

plant biology↗

bioRxiv2018

Determining targeting specificity of nuclear-encoded organelle proteins with the self-assembling split fluorescent protein toolkit

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo. Here, we have improved this technology with respect to sensitivity and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high throughput screening of candidate proteins we have developed a Golden Gate-based vector toolkit, named PlaMiNGo (Plastid and/or Mitochondria targeted proteins N-terminally fused to GFP11 tags via Golden Gate cloning). As a result of these improvements, dual targeting could be detected for a number of proteins, which had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach thus demonstrating the sensitivity and robustness of the sasplit-FP-based method to analyze the targeting specificity of nuclear-encoded proteins. HighlightSeveral mono-specific proteins showed dual targeting to plastids and mitochondria with the self-assembling split-GFP system. A Golden Gate-based vector toolkit was constructed to facilitate easy cloning and subsequent determination of protein targeting specificity.

plant biology↗

bioRxiv2018

PARbars: cheap, easy to build ceptometers for continuous measurement of light interception in plant canopies

Short AbstractDetailed instructions on how to build, calibrate and collect research quality data from PARbar ceptometers are presented.\n\nLong AbstractCeptometry is a technique used to measure the transmittance of photosynthetically active radiation through a plant canopy using multiple light sensors connected in parallel on a long bar. Ceptometry is often used to infer properties of canopy structure and light interception, notably leaf area index (LAI) and effective plant area index (PAIeff). Due to the high cost of commercially available ceptometers, the number of measurements that can be taken is often limited in space and time. This limits the usefulness of ceptometry for studying genetic variability in light interception, and precludes thorough analysis of, and correction for, biases that can skew measurements depending on the time of day. We developed continuously logging ceptometers (called PARbars) that can be produced for USD $75 each and yield high quality data comparable to commercially available alternatives. Here we provide detailed instruction on how to build and calibrate PARbars, how to deploy them in the field and how to estimate PAI from collected transmittance data. We provide representative results from wheat canopies and discuss further considerations that should be made when using PARbars.

plant biology↗

bioRxiv2018

High-throughput genotyping of the spelt gene pool reveals patterns of agricultural history in Europe

Spelt, a close relative of hexaploid bread wheat and a dominant wheat subspecies cultivated in Europe before the 20th century, still plays an important role as a high-value niche product today. Compared to most other cereals, spelt has not been subjected to intensive breeding in the 20th century. Even today, mostly traditional landraces are cultivated on a regional scale. The traditional way of spelt cultivation has limited the extensive exchange of germ plasm and intermixing of genetic material, which makes spelt an ideal crop to study the early agricultural history of cereals in Europe. Here, we unraveled the population structure and agricultural history of spelt based on 22,999 high-quality SNPs obtained by genotyping-by-sequencing on 267 spelt accessions covering the entire cultivation range. SNP markers were aligned to the high-quality bread wheat reference genome, which allowed us to analyze individual subgenomes. Our analyses of genetic variation revealed that bread wheat and spelt are most likely of monophyletic origin, but that European spelt diverged from bread wheat by hybridization with tetraploid emmer wheats. Interestingly, spelt accessions from the Iberian Peninsula formed a separate clade that was distinct from the Central European accessions for all three subgenomes. Demographic modelling indicated that Iberian spelt was introduced into Europe independently from Central European spelt. Our analysis provides a comprehensive assessment of spelt diversity and history. The separate introduction of Iberian spelt is supported by recent molecular evidence of two independent prehistoric migrations of ancient farmers from the Near East into Europe.

plant biology↗

bioRxiv2018

Epigenetics of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua

In plants, dioecy characterizes species carrying male and female flowers on separate plants and occurs in about 6% of angiosperms. To date, the molecular mechanism(s) underlying sexual dimorphism is essentially unknown. The ability of gender-reversal by hormone application suggests that epigenetics might play an important role in sexual dimorphism. Proteome analysis of nuclei derived from flower buds of females, males and feminized males of the dioecious plant Mercurialis annua revealed differentially expressed proteins related to nucleic acid binding proteins, hydrolases and transcription factors, including floral homeotic genes. Further analysis showed that class B genes are mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation could be found between the expression of class B and D genes and their DNA methylation or chromatin conformation. Thus, our results ruled out epigenetic control over floral identity genes as the major determinants regulating sexual dimorphisms. Rather, determination of sex in M. annua might be controlled upstream of floral identity genes by a gender-specific factor that affects hormonal homeostasis.\n\nHighlightsSex determination in Mercurialis annua is not related to epigenetics of floral homeotic genes but appears to be modulated by an unknown gender-specific regulator(s) that affects hormonal homeostasis.

plant biology↗

bioRxiv2018

Development of a New Method for the Highly Effective Identification of Cold Resistance in Living Avocado Varieties

This paper first identified the cold resistance of 38 varieties of avocado by determining the semi-lethal low temperature (LT50) of the leaves using an electrical conductivity method in combination with a logistic function, and then analyzed the correlation between the LT50 of 27 varieties and the capacitance measured 9 different parts of the leaves in vivo, to explore the relationship between the cold resistance of various avocado varieties and the capacitance of different parts of avocado leaves, so as to develop a new method for highly effective identification of cold resistance of living avocado varieties. The results showed that various avocado varieties LT50 was significantly positively correlated with the capacitance of some parts of leaves, showing that the cold resistance of various avocado varieties was negatively correlated with the capacitance of various leaf parts. The results of mango variety trials conducted for comparison is coincident with the theoretical conclusion reached in the identification of the cold resistance of the avocado. So as to the study of the cold resistance of avocado and mango varieties, the capacitance of live mature leaves measured in the field can be used as a new method for the judgment of cold resistance.\n\nHighlightA new method for identification of avocado cold resistance through measuring the capacitance of different parts of leaves was developed. This method are simple, quick and efficient.

plant biology↗

bioRxiv2018

Vulnerability to climate change for narrowly ranged species: the case of Ecuadorian endemic Magnolia mercedesiarum

Species vulnerability to climate change has been inferred using species distribution models from an example of the recently discovered Magnolia mercedesiarum (sect. Talauma, Magnoliaceae), a narrowly ranged species endemic to moist tropical forests in the eastern Ecuadorian Andes. The environmental conditions within the current species distribution area has been compared with conditions projected to 2050 and 2070, using data from the HadGEM2-ES model in two CO2 emission scenarios: RCP4.5 and RCP8.5. The ecological niche modelling allowed determination of parameters of climatic environmental conditions that control current species distribution to produce a hypothesis on probable changes in spatial pattern of suitable habitats in future scenarios. Within the current species distribution area of M. mercedesiarum, significant reduction of habitat suitability was projected for both emission scenarios, combined with a lack of nearby areas with adequate environmental conditions. Several disjunct sites of high habitat suitability were found to emerge in the Colombian Andes, but they seem unreachable by this tree species in the scope of a few decades, due to intrinsic dispersal limitations. The reduction of habitat suitability and improbability of distribution area shift to adjacent geographic locations could mean a high species vulnerability to climate change. The species could be at risk of extinction if it does not possess hidden phenotypical plasticity and potential for fast adaptation to climate change.

plant biology↗

bioRxiv2018

Subtilase-mediated activation of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway

Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and is a prerequisite for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilase SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis.

plant biology↗

bioRxiv2018

Effects of FLOWERING LOCUS T on FD during the transition to flowering at the shoot apical meristem of Arabidopsis thaliana

The transition to flowering is a crucial step in the plant life cycle that is controlled by multiple endogenous and environmental cues, including hormones, sugars, temperature, and photoperiod. Permissive photoperiod induces FLOWERING LOCUS T (FT) in the phloem companion cells of leaves. The FT protein then acts as a florigen that is transported to the shoot apical meristem (SAM) where it physically interacts with the bZIP transcription factor FD and 14-3-3 proteins. However, despite the importance of FD for promoting flowering, its direct transcriptional targets are largely unknown. Here we combined ChIP-seq and RNA-seq to identify targets of FD at the genome-wide scale and assess the contribution of FT to binding DNA. We further investigated the ability of FD to form protein complexes with FT and TFL1 through the interaction with 14-3-3 proteins. Importantly, we observe direct binding of FD to targets involved in several aspects of the plant development not directly related to the regulation of flowering time. Our results confirm FD as central regulator of the floral transition at the shoot meristem and provides evidence for crosstalk between the regulation of flowering and other signaling pathways.\n\nMaterial DistributionThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.cell.com/molecular-plant/authors): Markus Schmid (markus.schmid@umu.se).\n\nContact InformationUme[a] Plant Science Centre (UPSC), Dept. of Plant Physiology Ume[a] University, SE-901 87 Ume[a], SWEDEN

plant biology↗

bioRxiv2018

Engineering a decoy substrate in soybean to enable recognition of the Soybean Mosaic Virus NIa protease

In Arabidopsis, recognition of the AvrPphB effector protease from Pseudomonas syringae is mediated by the disease resistance (R) protein RPS5, which is activated by AvrPphB-induced cleavage of the Arabidopsis protein kinase PBS1. The recognition specificity of RPS5 can be altered by substituting the AvrPphB cleavage site within PBS1 with cleavage sequences for other proteases, including proteases from viruses. AvrPphB also activates defense responses in soybean (Glycine max), suggesting that soybean may contain an R protein analogous to RPS5. It was unknown, however, whether this response is mediated by cleavage of a soybean PBS1-like protein. Here we show that soybean contains three PBS1 orthologs and that their products are cleaved by AvrPphB. Further, transient expression of soybean PBS1 derivatives containing a five-alanine insertion at their AvrPphB cleavage sites activated cell death in soybean protoplasts, demonstrating that soybean likely contains an AvrPphB-specific resistance protein that is activated by a conformational change in soybean PBS1 proteins. Significantly, we show that a soybean PBS1 decoy protein modified to contain a cleavage site for the Soybean mosaic virus (SMV) NIa protease triggers cell death in soybean protoplasts when cleaved by this protease, indicating that the PBS1 decoy approach will work in soybean using endogenous PBS1 genes. Lastly, we show that activation of the AvrPphB-dependent cell death response effectively inhibits systemic spread of SMV in soybean. These data also indicate that decoy engineering may be feasible in other crop plant species that recognize AvrPphB protease activity.

plant biology↗

bioRxiv2018

Arbuscular cotton-associated mycorrhizal fungi in Yeola region of Maharashtra, India

Mycorrhizae are a mutual symbiotic link between the plant root and a fungus that colonizes the cortical tissue of the roots during active plant growth periods. Both the host plant and the fungus have the potential to benefit. Mycorrhizae are ubiquitous throughout the world in terrestrial ecosystems. The purpose of this study is to evaluate the association of arbuscular mycorrhizal fungi in cotton crops with AM fungal population density in rhizosphere soils, investigate the qualitative composition of AM fungal species and the percentage of root colonization. The results showed that the number of AM fungal propagules collected from different locations in cotton crops ranged from 235 to 1580 spores per 100 g of soil. Due to the widespread nature of AM fungi, they occurred in almost all soil samples, but the number and type of spores and sporocarps varied. In total, 41 AM fungal species belonging to the genera Glomus, Acaulospora and Scutellospora were isolated. Glomus was found to be predominantly followed by Scutellospora in cotton soils in the rhizosphere. The distribution of spores, density and composition of AM fungi are observed to be influenced by environmental and physicochemical factors. The AM spore number, root colonization percentage and distribution vary depending on the seasonal fluctuations in moisture, temperature, pH and soil mineral nutrient status such as OC, P2O5, K2O, Zn, Cu, Fe, Mn, etc. The obtained data shows that nitrogen-deficient soils had more AM fungal propagules. The soils with a high concentration of phosphorus and potassium had the least AM fungal spores. Depleted zinc, copper and manganese levels have also been positive for more fungal occurrence and distribution. The presence of high iron levels in the soil, however, encourages more AM spores and a percentage of root colonisation.

plant biology↗

bioRxiv2018

Plant-derived coumarins shape the composition of an Arabidopsis synthetic root microbiome

SignificanceThe root microbiome composition is largely determined by the soil inoculum, with a distinct contribution from the host. Yet, the molecular mechanisms with which the host influences its rhizobiome are only beginning to be discovered. Using a hydroponics-based synthetic community approach, we probe the impact of root-exuded specialized metabolites in shaping the root microbiome. We uncover a role for coumarins in structuring the rhizobiome, particularly by limiting the growth of a Pseudomonas strain, for which we propose a mechanism of action. Our findings support the exciting possibility that root-exuded coumarins form a part of the plants adaptive response to iron deficiency that goes beyond iron mobilization to modulate the rhizobiome, and highlights avenues towards engineering the rhizosphere for plant health.\n\nAbstractThe factors that dictate the composition of the root microbiome and its role in plant fitness has been a long-standing question. Recent work has highlighted a major contribution of the soil inoculum in determining the composition of the root microbiome. However, plants are known to conditionally exude a diverse array of unique secondary metabolites, largely varying between species and environmental conditions. Here, we explore the role of specialized metabolites in dictating which bacteria reside in the rhizosphere. We employed a reduced synthetic community (SynCom) of Arabidopsis thaliana root-isolated bacteria to detect community shifts that occur in the absence of the secreted small molecule phytoalexins, flavonoids, and coumarins. We find that lack of coumarin biosynthesis in f6h1 mutant plant lines causes a shift in the root microbial community specifically under iron deficiency. We demonstrate a potential role for iron-mobilizing coumarins in sculpting the A. thaliana root bacterial community by inhibiting the proliferation of a relatively abundant Pseudomonas species via a redox-mediated mechanism. This work establishes a systematic approach enabling elucidation of specific mechanisms by which plant-derived molecules mediate microbial community composition. Our findings expand on the function of conditionally-exuded specialized metabolites and lead to new avenues to effectively engineer the rhizosphere for improving crop growth in alkaline soils, which make up a third of total arable soils.

plant biology↗

bioRxiv2018

Differential responses of antioxidants and dehydrin expression in two switchgrass (Panicum virgatum) cultivars contrasting in drought tolerance

Drought stress is a major limiting factor for plant growth and development in many regions of the world. This study was designed to investigate antioxidant metabolism and dehydrin expression responses to drought stress in two switchgrass cultivars (drought tolerant Alamo, and drought sensitive Dacotah) contrasting in drought tolerance. The plants were subjected to well-watered [100% evapotranspiration (ET)] or drought stress (30%-50% ET) conditions for up to 24 d in growth chambers. Drought stress decreased leaf relative water content (RWC), increased leaf electrolyte leakage (EL), leaf malondialdehyde (MDA) content in two cultivars, but Alamo exhibited higher leaf RWC level, lower leaf EL and MDA when compared to Dacotah at 24 d of drought treatment. Drought stress also increased superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities in two cultivars, Alamo had relatively higher SOD, CAT and APX activities and greater abundance of SOD and APX isozymes than Dacotah at 24 d of drought treatment. Alamo had higher abundance of 55 KDa and 18 KDa dehydrin accumulation than Dacotah under drought treatment. Relative genes expression level of PvCAT1, PvAPX2, PvERD and PvPIP1;5 in Alamo were significantly higher than Dacotah at 24 d of drought treatment. These results suggest that increase in antioxidant enzymes and accumulation of dehydrin were highly related with switchgrass drought tolerance. Antioxidant enzyme activity, isozyme expression and dehydrin abundance could provide a useful screening tool to identify relative drought tolerance in switchgrass cultivars.

plant biology↗

bioRxiv2018

Pheophorbide a, a chlorophyll catabolite may regulate jasmonate signalling during dark-induced senescence in Arabidopsis

Chlorophyll degradation is one of the most visible landmarks of leaf senescence. During senescence, chlorophyll is degraded in the multi-step pheophorbide a oxygenase (PAO)/phyllobilin pathway, which is tightly regulated at the transcriptional level. This regulation allows a coordinated and efficient remobilisation of nitrogen towards sink organs. Taking advantage of combined transcriptome and metabolite analyses during dark-induced senescence of Arabidopsis thaliana mutants deficient in key steps of the PAO/phyllobilin pathway, we show an unanticipated role for one of the pathway intermediates, i.e. pheophorbide a. Both jasmonic acid-related gene expression and jasmonic acid precursors specifically accumulated in pao1, deficient in PAO. We propose that pheophorbide a, the last intact porphyrin intermediate of chlorophyll degradation and unique pathway bottleneck, has been recruited as a signalling molecule of the chloroplast metabolic status. Our work challenges the assumption that chlorophyll breakdown is merely a senescence output, but propose that the flux of pheophorbide a through the pathway acts in a feed-forward loop that remodels the nuclear transcriptome and controls the pace of chlorophyll degradation in senescing leaves.\n\nSummaryTranscriptome and metabolite profiles of key chlorophyll breakdown mutants reveal complex interplay between speed of chlorophyll degradation and jasmonic acid signalling\n\nFinancial sourcesThis work was supported by the European Union (Plant Fellow program), the Swiss National Foundation/ERA-NET (grant N{degrees} 163504) and the German Research Foundation (DFG, grant N{degrees} INST 186/822-1).

plant biology↗

bioRxiv2018

CRK2 enhances salt tolerance in Arabidopsis thaliana by regulating endocytosis and callose deposition in connection with PLDα1

High salinity has become an increasingly prevalent source of stress to which plants need to adapt. The receptor-like protein kinases (RLKs), including the cysteine-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants and are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses, however their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis thaliana. We identified CRK2 as a negative regulator of endocytosis, under both normal growth conditions and salt stress. We also established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a novel role for callose deposition, in response to increased salinity, and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins we observed specific changes in CRK2s subcellular localization in response to various stress treatments. Many of CRK2s cellular functions were dependent on phospholipase D (PLD) activity, as were the subcellular localization changes. Thus we propose that CRK2 acts downstream of PLD during salt stress to regulate endocytosis and promote callose deposition, and that CRK2 adopts specific stress-dependent subcellular localization patterns in order to carry out its functions.\n\nOne sentence summaryThe receptor-like kinase CRK2 acts in connection with PLD1 to regulate endocytosis and callose deposition at plasmodesmata, enhancing salt tolerance in Arabidopsis thaliana.

plant biology↗

bioRxiv2018

Antiviral Functions of ARGONAUTE Proteins During Turnip Crinkle Virus Infection Revealed by Image-based Trait Analysis in Arabidopsis

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted of by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green and blue (RGB) imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent non-additive, anti-TCV effectors, while AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.\n\nAuthor SummaryPlant viruses caused substantial losses in crop production and quality worldwide. Precisely measuring plant health is critical for better understanding the mechanisms underlying plant virus and host interactions. Advances in high-resolution imaging technologies and deep-learning tools have made acquiring and analyzing \"big data\" of disease traits possible. In this study, we have developed a high-throughput, image-based trait phenotyping pipeline to quantify disease severity in Arabidopsis thaliana infected by Turnip Crinkle Virus (TCV). Our aim is to understand how the antiviral RNA silencing machinery is tuned to protect the host from invading virus infection. We focused on ARGONAUTE proteins, which are the effectors in the RNA silencing pathway. A mutant line of TCV with a dysfunctional silencing suppressor (P38) was used to investigate which ago mutation could compensate for the dysfunctional silencing suppressor and facilitate the development of disease symptoms. We demonstrated that specific AGO proteins contribute to protecting leaves from TCV infection in a non-additive manner. Our results also implied that distinct AGOs are required to function collectively to silence TCV in inflorescence tissues. More evidence is still needed to further understand how these antiviral AGOs interact with suppressor proteins molecularly during TCV infection.

plant biology↗

bioRxiv2018

Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum.

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the golden-coloured marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.\n\nHighlightWe discover RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum and show that they have surprising functional and structural features, and belong to novel red-plastid lineage RSH clades.

plant biology↗

bioRxiv2018

Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL1, MSL2, and MSL3 reveal a role for inter-organellar communication in plant development

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between three Mechanosensitive channels of Small conductance-Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bacterial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to the inner mitochondrial membrane, while MSL2 and MSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion in MSL1, both in wild type and in msl2 msl3 mutant backgrounds. msl1 single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling in msl2 msl3 double mutants was exacerbated in the msl1 msl2 msl3 triple mutant. However, the introduction of the msl1 lesion into the msl2 msl3 mutant background suppressed other msl2 msl3 mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version of MSL1. In yeast-based interaction studies, MSL1 interacted with itself, but not with MSL2 or MSL3. These results establish that the abnormalities observed in msl2 msl3 double mutants is partially dependent on the presence of functional MSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

plant biology↗