Hybrid cyanobacterial-tobacco Rubisco supports autotrophic growth and pre-carboxysomal aggregation

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO2 fixing enzyme Rubisco. Cyanobacteria utilize a CO2 concentrating mechanism (CCM), which encapsulates Rubisco with poor specificity but a relatively fast catalytic rate within a carboxysome micro-compartment. Alongside the active transport of bicarbonate into the cell, and localization of carbonic anhydrase within the carboxysome shell with Rubisco, cyanobacteria are able to overcome the limitations of Rubisco via localization within a high CO2 environment. As part of ongoing efforts to engineer a β-cyanobacterial CCM into land plants, we investigated the potential for Rubisco large subunits (LSU) from the β-cyanobacteria Synechococcus elongatus (Se) to form aggregated Rubisco complexes with the carboxysome linker protein CcmM35 within tobacco (Nicotiana tabacum) chloroplasts. Transplastomic plants were produced that lacked cognate SeRubisco small subunits (SSU) and expressed SeLSU in place of tobacco LSU, with and without CcmM35. Plants were able to form a hybrid enzyme utilizing tobacco SSU and the SeLSU, allowing slow autotrophic growth in high CO2. CcmM35 was able to form large Rubisco aggregates with the SeLSU, and these incorporated small amounts of native tobacco SSU. Plants lacking the SeSSU showed delayed growth, poor photosynthetic capacity and significantly reduced Rubisco activity compared to both wild-type tobacco and lines expressing the SeSSU. These results demonstrate the ability of the SeLSU and CcmM35 to form large aggregates without the cognate SeSSU in planta, harboring active Rubisco that enables plant growth, albeit at a much slower pace than plants expressing the cognate SeSSU

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants

Photosynthetic inefficiencies limit the productivity and sustainability of crop production, and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest to improve the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid’s efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization, the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modifying rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes

Jasmonic acid-dependent regulation of seed dormancy following maternal herbivory in Arabidopsis

Maternal experience of abiotic environmental factors such as temperature and light are well known to control seed dormancy in many plant species. Maternal biotic stress alters offspring defence phenotypes, but whether it also affects seed dormancy remains unexplored. We exposed Arabidopsis thaliana plants to herbivory and investigated plasticity in germination and defence phenotypes in their offspring, along with the roles of phytohormone signalling in regulating maternal effects. Maternal herbivory resulted in the accumulation of jasmonic acid-isoleucine and loss of dormancy in seeds of stressed plants. Dormancy was also reduced by engineering seed-specific accumulation of jasmonic acid in transgenic plants. Loss of dormancy was dependent on an intact jasmonate signalling pathway and was associated with increased gibberellin content and reduced abscisic acid sensitivity during germination. Altered dormancy was only observed in the first generation following herbivory, whereas defence priming was maintained for at least two generations. Herbivory generates a jasmonic acid-dependent reduction in seed dormancy, mediated by alteration of gibberellin and abscisic acid signalling. This is a direct maternal effect, operating independently from transgenerational herbivore resistance priming

Burden of Risk Alleles for Hypertension Increases Risk of Intracerebral Hemorrhage

Background and Purpose-Genetic variation influences risk of intracerebral hemorrhage (ICH). Hypertension (HTN) is a potent risk factor for ICH and several common genetic variants (single nucleotide polymorphisms [SNPs]) associated with blood pressure levels have been identified. We sought to determine whether the cumulative burden of blood pressure-related SNPs is associated with risk of ICH and pre-ICH diagnosis of HTN. Methods-We conducted a prospective multicenter case-control study in 2272 subjects of European ancestry (1025 cases and 1247 control subjects). Thirty-nine SNPs reported to be associated with blood pressure levels were identified from the National Human Genome Research Institute genomewide association study catalog. Single-SNP association analyses were performed for the outcomes ICH and pre-ICH HTN. Subsequently, weighted and unweighted genetic risk scores were constructed using these SNPs and entered as the independent variable in logistic regression models with ICH and pre-ICH HTN as the dependent variables. Results-No single SNP was associated with either ICH or pre-ICH HTN. The blood pressure-based unweighted genetic risk score was associated with risk of ICH (OR, 1.11; 95% CI, 1.02-1.21; P=0.01) and the subset of ICH in deep regions (OR, 1.18; 95% CI, 1.07-1.30; P=0.001), but not with the subset of lobar ICH. The score was associated with a history of HTN among control subjects (OR, 1.17; 95% CI, 1.04-1.31; P=0.009) and ICH cases (OR, 1.15; 95% CI, 1.01-1.31; P=0.04). Similar results were obtained when using a weighted score. Conclusion-Increasing numbers of high blood pressure-related alleles are associated with increased risk of deep ICH as well as with clinically identified HTN. (Stroke. 2012; 43: 2877-2883.

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

The role of callose and beta(1,3)-glucanase in microsporogenesis.

The significance of the tetrad callose wall and its hydrolysis at microspore release has been investigated in Brassica napus. Callose wall breakdown was analyzed by measuring activities of beta(1,3)-glucanase enzymes in relation to different stages of anther development. Assaying B. napus buds for endolytic and exolytic beta(1,3)-glucanases established that activity peaked at the time of callose dissolution. Initial experiments to clone genes encoding these enzymes involved the use of probes related to PR beta(1,3)-glucanases. Sequencing of an anther-specific cDNA, A6, which was isolated from a B. napus 'sporogenesis' library, revealed similarity to previously characterized endo-beta(1,3)- and beta(1,3;1,4)-glucanases. Polyclonal antibody raised to the A6 protein identified a temporally-regulated ~60 kD band in B. napus buds which attained maximum levels at microspore release stage. Further analysis revealed that this ~60 kD band represented several proteins of different charge but similar molecular weight. A6 thus appears to be part of a family of immunologically related proteins. The sequence data and the temporal expression pattern suggest that A6 may be part of the callase enzyme complex involved in microspore release. The role of the callose wall was investigated by engineering its premature removal in vivo. This was achieved by expressing a modified PR beta(1,3)-glucanase in the anther tapetum of transgenic Nicotiana tabacum. These transgenic plants exhibited reduced male fertility, ranging from partial to complete male sterility. The microspores from the reduced fertility transgenic plants had an abnormal exine and the tapetum demonstrated hypertrophy. Male sterility appeared to result from bursting of the aberrant microspores after microspore release. These results suggested that premature callose hydrolysis is sufficient to cause male sterility and that callose is involved in exine formation in N. tabacum

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase

The regulation of Rubisco, the gatekeeper of carbon fixation into the biosphere, by its molecular chaperone Rubisco activase (Rca) is essential for photosynthesis and plant growth. Using energy from ATP hydrolysis, Rca promotes the release of inhibitors and restores catalytic competence to Rubisco-active sites. Rca is sensitive to moderate heat stress, however, and becomes progressively inhibited as the temperature increases above the optimum for photosynthesis. Here, we identify a single amino acid substitution (M159I) that fundamentally alters the thermal and regulatory properties of Rca in bread wheat (Triticum aestivum L.). Using site-directed mutagenesis, we demonstrate that the M159I substitution extends the temperature optimum of the most abundant Rca isoform by 5°C in vitro, while maintaining the efficiency of Rubisco activation by Rca. The results suggest that this single amino acid substitution acts as a thermal and regulatory switch in wheat Rca that can be exploited to improve the climate resilience and efficiency of carbon assimilation of this cereal crop as temperatures become warmer and more volatile

Sphingolipids, new players in plant signalling.

Sphingolipids are a diverse group of compounds, some of which play important signaling roles in animals and yeast. Results from recent research suggest that not only do plants contain components present in animal sphingolipid signaling pathways but that they might also possess novel plant-specific sphingolipid signaling systems. We suggest that the time is ripe for an in depth investigation of the role of this enigmatic group of compounds in plants

Rubisco activation by wheat Rubisco activase isoform 2β is insensitive to inhibition by ADP

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and ADP/ATP ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1β (42.7 kDa), 2β (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2β was insensitive to ADP inhibition, while Rca 1β and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1β and 2β could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2β to the corresponding residues in 1β (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO2 assimilation

Treating seeds with activators of plant defence generates long-lasting priming of resistance to pests and pathogens.

Priming of defence is a strategy employed by plants exposed to stress to enhance resistance against future stress episodes with minimal associated costs on growth. Here, we test the hypothesis that application of priming agents to seeds can result in plants with primed defences. We measured resistance to arthropod herbivores and disease in tomato (Solanum lycopersicum) plants grown from seed treated with jasmonic acid (JA) and/or beta-aminobutryric acid (BABA). Plants grown from JA-treated seed showed increased resistance against herbivory by spider mites, caterpillars and aphids, and against the necrotrophic fungal pathogen, Botrytis cinerea. BABA seed treatment provided primed defence against powdery mildew disease caused by the biotrophic fungal pathogen, Oidium neolycopersici. Priming responses were long-lasting, with significant increases in resistance sustained in plants grown from treated seed for at least eight weeks, and were associated with enhanced defence gene expression during pathogen attack. There was no significant antagonism between different forms of defence in plants grown from seeds treated with a combination of JA and BABA. Long-term defence priming by seed treatments was not accompanied by reductions in growth, and may therefore be suitable for commercial exploitation