Environmental conditions profoundly affect plant disease development; however, the underlying molecular bases are not well understood. Here we show that elevated temperature significantly increases the susceptibility of Arabidopsis to Pseudomonas syringae pv. tomato (Pst) DC3000 independently of the phyB/PIF thermosensing pathway. Instead, elevated temperature promotes translocation of bacterial effector proteins into plant cells and causes a loss of ICS1-mediated salicylic acid (SA) biosynthesis. Global transcriptome analysis reveals a major temperature-sensitive node of SA signalling, impacting ~60% of benzothiadiazole (BTH)-regulated genes, including ICS1 and the canonical SA marker gene, PR1. Remarkably, BTH can effectively protect Arabidopsis against Pst DC3000 infection at elevated temperature despite the lack of ICS1 and PR1 expression. Our results highlight the broad impact of a major climate condition on the enigmatic molecular interplay between temperature, SA defence and function of a central bacterial virulence system in the context of a widely studied susceptible plant-pathogen interaction.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Since they emerged approximately 125 million years ago, flowering plants have evolved to dominate the terrestrial landscape and survive in the most inhospitable environments on earth. At their core, these adaptations have been shaped by changes in numerous, interconnected pathways and genes that collectively give rise to emergent biological phenomena. Linking gene expression to morphological outcomes remains a grand challenge in biology, and new approaches are needed to begin to address this gap. Here, we implemented topological data analysis (TDA) to summarize the high dimensionality and noisiness of gene expression data using lens functions that delineate plant tissue and stress responses. Using this framework, we created a topological representation of the shape of gene expression across plant evolution, development, and environment for the phylogenetically diverse flowering plants. The TDA-based Mapper graphs form a well-defined gradient of tissues from leaves to seeds, or from healthy to stressed samples, depending on the lens function. This suggests that there are distinct and conserved expression patterns across angiosperms that delineate different tissue types or responses to biotic and abiotic stresses. Genes that correlate with the tissue lens function are enriched in central processes such as photosynthetic, growth and development, housekeeping, or stress responses. Together, our results highlight the power of TDA for analyzing complex biological data and reveal a core expression backbone that defines plant form and function.

The Helical Carotenoid Proteins (HCPs) are a large group of newly identified carotenoid-binding proteins found in ecophysiologically diverse cyanobacteria. They likely evolved before becoming the effector (quenching) domain of the modular Orange Carotenoid Protein (OCP). The number of discrete HCP families-at least nine-suggests they are involved in multiple distinct functions. Here we report the 1.7 Å crystal structure of HCP2, one of the most widespread HCPs found in nature, from the chromatically acclimating cyanobacterium Tolypothrix sp. PCC 7601. By purifying HCP2 from the native source we are able to identify its natively-bound carotenoid, which is exclusively canthaxanthin. In solution, HCP2 is a monomer with an absorbance maximum of 530 nm. However, the HCP2 crystals have a maximum absorbance at 548 nm, which is accounted by the stacking of the β1 rings of the carotenoid in the two molecules in the asymmetric unit. Our results demonstrate how HCPs provide a valuable system to study carotenoid-protein interactions and their spectroscopic implications, and contribute to efforts to understand the functional roles of this large, newly discovered family of pigment proteins, which to-date remain enigmatic.

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.

The tryptophan-rich sensory protein (TSPO) is a membrane protein, which is a member of the 18 kDa translocator protein/peripheral-type benzodiazepine receptor (MBR) family of proteins that is present in most organisms and is also referred to as Translocator protein 18 kDa. Although TSPO is associated with stress- and disease-related processes in organisms from bacteria to mammals, full elucidation of the functional role of the TSPO protein is lacking for most organisms in which it is found. In this study, we describe the regulation and function of a TSPO homolog in the cyanobacterium Fremyella diplosiphon, designated FdTSPO. Accumulation of the FdTSPO transcript is upregulated by green light and in response to nutrient deficiency and stress. A F. diplosiphon TSPO deletion mutant (i.e., ΔFdTSPO) showed altered responses compared to the wild type (WT) strain under stress conditions, including salt treatment, osmotic stress, and induced oxidative stress. Under salt stress, the FdTSPO transcript is upregulated and a ΔFdTSPO mutant accumulates lower levels of reactive oxygen species (ROS) and displays increased growth compared to WT. In response to osmotic stress, FdTSPO transcript levels are upregulated and ΔFdTSPO mutant cells exhibit impaired growth compared to the WT. By comparison, methyl viologen-induced oxidative stress results in higher ROS levels in the ΔFdTSPO mutant compared to the WT strain. Taken together, our results provide support for the involvement of membrane-localized FdTSPO in mediating cellular responses to stress in F. diplosiphon and represent detailed functional analysis of a cyanobacterial TSPO. This study advances our understanding of the functional roles of TSPO homologs in vivo.

Light-dependent modification of photosynthetic pigmentation and cellular growth responses is commonly associated with increased fitness in photosynthetic organisms, including cyanobacteria. Prior analyses of pigmentation mutants in the freshwater cyanobacterium Fremyelladiplosiphon has resulted in the observation that RcaE is a photosensor responsible for regulating organismal responses to changes in red light (RL) and green light (GL). RcaE regulates both pigmentation and cellular morphology, yet previous investigations and the analysis of additional pigmentation mutants here show that the signaling pathways regulating pigmentation and morphology appear to branch downstream of RcaE. We provide evidence that a ΔcpeR mutant has altered regulation of cellular morphology in addition to a known disruption in phycoerythrin synthesis. This marks the first description of the association of a regulator with the control of cellular morphology under both RL and GL in F.diplosiphon, apart from RcaE. In addition to providing a link between CpeR and the photoregulation of morphology in F.diplosiphon, the isolation of a ΔcpeR::IS66 mutant in the UTEX 481 strain represents both the first isolation of an IS66-based gene disruption and verification of the existence of an IS66-related element in F. diplosiphon.

Circadian rhythm pathways influence the expression patterns of as much as 31% of the Arabidopsis genome through complicated interaction pathways, and have been found to be significantly disrupted by biotic and abiotic stress treatments, complicating treatment-response gene discovery methods due to clock pattern mismatches in the fold change-based statistics. The PRIISM (Pattern Recomposition for the Isolation of Independent Signals in Microarray data) algorithm outlined in this paper is designed to separate pattern changes induced by different forces, including treatment-response pathways and circadian clock rhythm disruptions.

Chloroplast-localized sigma factor (SIG) proteins promote specificity of the plastid-encoded RNA polymerase. SIG2 function appears to be necessary for light-grown Arabidopsis thaliana plants. Specific photoreceptors or light-dependent factors that impact the light-induced accumulation of SIG2 have not been reported. A molecular link between phytochromes and nuclear-encoded SIG2, which impacts photomorphogenesis specifically under red (R) and far-red (FR) light, is described here. Both phyA and phyB promote SIG2 transcript accumulation. Disruption of SIG2 results in R- and FR-specific defects in the inhibition of hypocotyl elongation and cotyledon expansion, although no impairments in these responses are detected for sig2 mutants under blue (B) or white (W) light. SIG2 also impacts root elongation under W and R, and the R-dependent expression of PIF4, encoding a phytochrome-interacting factor, and HY2, which encodes a phytochrome chromophore biosynthetic enzyme. Whereas SIG2 apparently impacts the accumulation of the phytochromobilin (PΦB) phytochrome chromophore, sig2 mutants differ significantly from PΦB mutants, primarily due to wavelength-specific defects in photomorphogenesis and disruption of a distinct subset of phytochrome-dependent responses. The molecular link between phytochromes and SIG2 is likely to be an important part of the co-ordination of gene expression to maintain stoichiometry between the nuclear-encoded phytochrome apoprotein and plastid-derived PΦB, which combine to form photoactive phytochromes, and/or light-dependent SIG2 accumulation is involved in an inductive light signalling pathway co-ordinating components between nucleus and plastids.

Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors-outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo.

Two proteins found in cyanobacteria contain a C-terminal domain with homology to the small subunit of rubisco (RbcS). These small subunit-like domains (SSLDs) are important features of CcmM, a protein involved in the biogenesis of carboxysomes found in all β-cyanobacteria, and a rubisco activase homolog [activase-like protein of cyanobacteria (ALC)] found in over a third of sequenced cyanobacterial genomes. Interaction with rubisco is crucial to the function of CcmM and is believed to be important to ALC as well. In both cases, the SSLD aggregates rubisco, and this nucleation event may be important in regulating rubisco assembly and activity. Recently, two independent studies supported the conclusion that the SSLD of CcmM binds equatorially to L8S8 holoenzymes of rubisco rather than by displacing an RbcS, as its structural homology would suggest. We use sequence analysis and homology modeling to examine whether the SSLD from the ALC could bind the large subunit of rubisco either via an equatorial interaction or in an RbcS site, if available. We suggest that the SSLD from the ALC of Fremyella diplosiphon could bind either in a vacant RbcS site or equatorially. Our homology modeling takes into account N-terminal residues not represented in available cryo-electron microscopy structures that potentially contribute to the interface between the large subunit of rubisco (RbcL) and RbcS. Here, we suggest the perspective that binding site variability as a means of regulation is plausible and that the dynamic interaction between the RbcL, RbcS, and SSLDs may be important for carboxysome assembly and function.

Carboxysomes are central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation in cyanobacteria. Although the structure is well understood, roles of environmental cues in the synthesis, positioning, and functional tuning of carboxysomes have not been systematically studied. Fremyella diplosiphon is a model cyanobacterium for assessing impacts of environmental light cues on photosynthetic pigmentation and tuning of photosynthetic efficiency during complementary chromatic acclimation (CCA), which is controlled by the photoreceptor RcaE. Given the central role of carboxysomes in photosynthesis, we investigated roles of light-dependent RcaE signaling in carboxysome structure and function. A ΔrcaE mutant exhibits altered carboxysome size and number, ccm gene expression, and carboxysome protein accumulation relative to the wild-type (WT) strain. Several Ccm proteins, including carboxysome shell proteins and core-nucleating factors, overaccumulate in ΔrcaE cells relative to WT cells. Additionally, levels of carboxysome cargo RuBisCO in the ΔrcaE mutant are lower than or unchanged from those in the WT strain. This shift in the ratios of carboxysome shell and nucleating components to the carboxysome cargo appears to drive carboxysome morphology and abundance dynamics. Carboxysomes are also occasionally mislocalized spatially to the periphery of spherical mutants within thylakoid membranes, suggesting that carboxysome positioning is impacted by cell shape. The RcaE photoreceptor links perception of external light cues to regulating carboxysome structure and function and, thus, to the cellular capacity for carbon fixation. IMPORTANCE Carboxysomes are proteinaceous subcellular compartments, or bacterial organelles, found in cyanobacteria that consist of a protein shell surrounding a core primarily composed of the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) that is central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation. Whereas significant insights have been gained regarding the structure and synthesis of carboxysomes, limited attention has been given to how their size, abundance, and protein composition are regulated to ensure optimal carbon fixation in dynamic environments. Given the centrality of carboxysomes in photosynthesis, we provide an analysis of the role of a photoreceptor, RcaE, which functions in matching photosynthetic pigmentation to the external environment during complementary chromatic acclimation and thereby optimizing photosynthetic efficiency, in regulating carboxysome dynamics. Our data highlight a role for RcaE in perceiving external light cues and regulating carboxysome structure and function and, thus, in the cellular capacity for carbon fixation and organismal fitness.

Second messengers are intracellular molecules regulated by external stimuli known as first messengers that are used for rapid organismal responses to dynamic environmental changes. Cyclic di-AMP (c-di-AMP) is a relatively newly discovered second messenger implicated in cell wall homeostasis in many pathogenic bacteria. C-di-AMP is synthesized from ATP by diadenylyl cyclases (DAC) and degraded by specific c-di-AMP phosphodiesterases (PDE). C-di-AMP DACs and PDEs are present in all sequenced cyanobacteria, suggesting roles for c-di-AMP in the physiology and/or development of these organisms. Despite conservation of these genes across numerous cyanobacteria, the functional roles of c-di-AMP in cyanobacteria have not been well-investigated. In a unique feature of cyanobacteria, phylogenetic analysis indicated that the broadly conserved DAC, related to CdaA/DacA, is always co-associated in an operon with genes critical for controlling cell wall synthesis. To investigate phenotypes regulated by c-di-AMP in cyanobacteria, we overexpressed native DAC (sll0505) and c-di-AMP PDE (slr0104) genes in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) to increase and decrease intracellular c-di-AMP levels, respectively. DAC- and PDE-overexpression strains, showed abnormal aggregation phenotypes, suggesting functional roles for regulating c-di-AMP homeostasis in vivo. As c-di-AMP may be implicated in osmotic responses in cyanobacteria, we tested whether sorbitol and NaCl stresses impacted expression of sll0505 and slr0104 or intracellular c-di-AMP levels in Synechocystis. Additionally, to determine the range of cyanobacteria in which c-di-AMP may function, we assessed c-di-AMP levels in two unicellular cyanobacteria, i.e., Synechocystis and Synechococcus elongatus PCC 7942, and two filamentous cyanobacteria, i.e., Fremyella diplosiphon and Anabaena sp. PCC 7120. C-di-AMP levels responded differently to abiotic stress signals in distinct cyanobacteria strains, whereas salt stress uniformly impacted another second messenger cyclic di-GMP in cyanobacteria. Together, these results suggest regulation of c-di-AMP homeostasis in cyanobacteria and implicate a role for the second messenger in maintaining cellular fitness in response to abiotic stress.

Cyanobacteria use a carbon dioxide (CO2)-concentrating mechanism (CCM) that enhances their carbon fixation efficiency and is regulated by many environmental factors that impact photosynthesis, including carbon availability, light levels, and nutrient access. Efforts to connect the regulation of the CCM by these factors to functional effects on carbon assimilation rates have been complicated by the aqueous nature of cyanobacteria. Here, we describe the use of cyanobacteria in a semiwet state on glass fiber filtration discs-cyanobacterial discs-to establish dynamic carbon assimilation behavior using gas exchange analysis. In combination with quantitative PCR (qPCR) and transmission electron microscopy (TEM) analyses, we linked the regulation of CCM components to corresponding carbon assimilation behavior in the freshwater, filamentous cyanobacterium Fremyella diplosiphon Inorganic carbon (Ci) levels, light quantity, and light quality have all been shown to influence carbon assimilation behavior in F. diplosiphon Our results suggest a biphasic model of cyanobacterial carbon fixation. While behavior at low levels of CO2 is driven mainly by the Ci uptake ability of the cyanobacterium, at higher CO2 levels, carbon assimilation behavior is multifaceted and depends on Ci availability, carboxysome morphology, linear electron flow, and cell shape. Carbon response curves (CRCs) generated via gas exchange analysis enable rapid examination of CO2 assimilation behavior in cyanobacteria and can be used for cells grown under distinct conditions to provide insight into how CO2 assimilation correlates with the regulation of critical cellular functions, such as the environmental control of the CCM and downstream photosynthetic capacity.IMPORTANCE Environmental regulation of photosynthesis in cyanobacteria enhances organismal fitness, light capture, and associated carbon fixation under dynamic conditions. Concentration of carbon dioxide (CO2) near the carbon-fixing enzyme RubisCO occurs via the CO2-concentrating mechanism (CCM). The CCM is also tuned in response to carbon availability, light quality or levels, or nutrient access-cues that also impact photosynthesis. We adapted dynamic gas exchange methods generally used with plants to investigate environmental regulation of the CCM and carbon fixation capacity using glass fiber-filtered cells of the cyanobacterium Fremyella diplosiphon We describe a breakthrough in measuring real-time carbon uptake and associated assimilation capacity for cells grown in distinct conditions (i.e., light quality, light quantity, or carbon status). These measurements demonstrate that the CCM modulates carbon uptake and assimilation under low-Ci conditions and that light-dependent regulation of pigmentation, cell shape, and downstream stages of carbon fixation are critical for tuning carbon uptake and assimilation.

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs - two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.