The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts.

A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

PhenoMeter: A Metabolome Database Search Tool Using Statistical Similarity Matching of Metabolic Phenotypes for High-Confidence Detection of Functional Links.

This article describes PhenoMeter (PM), a new type of metabolomics database search that accepts metabolite response patterns as queries and searches the MetaPhen database of reference patterns for responses that are statistically significantly similar or inverse for the purposes of detecting functional links. To identify a similarity measure that would detect functional links as reliably as possible, we compared the performance of four statistics in correctly top-matching metabolic phenotypes of Arabidopsis thaliana metabolism mutants affected in different steps of the photorespiration metabolic pathway to reference phenotypes of mutants affected in the same enzymes by independent mutations. The best performing statistic, the PM score, was a function of both Pearson correlation and Fisher's Exact Test of directional overlap. This statistic outperformed Pearson correlation, biweight midcorrelation and Fisher's Exact Test used alone. To demonstrate general applicability, we show that the PM reliably retrieved the most closely functionally linked response in the database when queried with responses to a wide variety of environmental and genetic perturbations. Attempts to match metabolic phenotypes between independent studies were met with varying success and possible reasons for this are discussed. Overall, our results suggest that integration of pattern-based search tools into metabolomics databases will aid functional annotation of newly recorded metabolic phenotypes analogously to the way sequence similarity search algorithms have aided the functional annotation of genes and proteins. PM is freely available at MetabolomeExpress (https://www.metabolome-express.org/phenometer.php).

Temperature response of in vivo Rubisco kinetics and mesophyll conductance in Arabidopsis thaliana: comparisons to Nicotiana tabacum.

Biochemical models are used to predict and understand the response of photosynthesis to rising temperatures and CO2 partial pressures. These models require the temperature dependency of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics and mesophyll conductance to CO2 (g(m)). However, it is not known how the temperature response of Rubisco kinetics differs between species, and comprehensive in vivo Rubisco kinetics that include gm have only been determined in the warm-adapted Nicotiana tabacum. Here, we measured the temperature response of Rubisco kinetics and gm in N. tabacum and the cold-adapted Arabidopsis thaliana using gas exchange and (13)CO2 isotopic discrimination on plants with genetically reduced levels of Rubisco. While the individual Rubisco kinetic parameters in N. tabacum and A. thaliana were similar across temperatures, they collectively resulted in significantly different modelled rates of photosynthesis. Additionally, gm increased with temperature in N. tabacum but not in A. thaliana. These findings highlight the importance of considering species-dependent differences in Rubisco kinetics and gm when modelling the temperature response of photosynthesis.

Antisense reductions in the PsbO protein of photosystem II leads to decreased quantum yield but similar maximal photosynthetic rates.

Photosystem (PS) II is the multisubunit complex which uses light energy to split water, providing the reducing equivalents needed for photosynthesis. The complex is susceptible to damage from environmental stresses such as excess excitation energy and high temperature. This research investigated the in vivo photosynthetic consequences of impairments to PSII in Arabidopsis thaliana (ecotype Columbia) expressing an antisense construct to the PsbO proteins of PSII. Transgenic lines were obtained with between 25 and 60% of wild-type (WT) total PsbO protein content, with the PsbO1 isoform being more strongly reduced than PsbO2. These changes coincided with a decrease in functional PSII content. Low PsbO (less than 50% WT) plants grew more slowly and had lower chlorophyll content per leaf area. There was no change in content per unit area of cytochrome b6f, ATP synthase, or Rubisco, whereas PSI decreased in proportion to the reduction in chlorophyll content. The irradiance response of photosynthetic oxygen evolution showed that low PsbO plants had a reduced quantum yield, but matched the oxygen evolution rates of WT plants at saturating irradiance. It is suggested that these plants had a smaller pool of PSII centres, which are inefficiently connected to antenna pigments resulting in reduced photochemical efficiency.

Chlorophyll fluorescence screening of Arabidopsis thaliana for CO2 sensitive photorespiration and photoinhibition mutants.

Exposure of Arabidopsis thaliana (L.) photorespiration mutants to air leads to a rapid decline in the Fv/Fm chlorophyll fluorescence parameter, reflecting a decline in PSII function and an onset of photoinhibition. This paper demonstrates that chlorophyll fluorescence imaging of Fv/Fm can be used as an easy and efficient means of detecting Arabidopsis mutants that are impaired in various aspects of photorespiration. This screen was developed to be sensitive and high throughput by the use of exposure to zero CO2 conditions and the use of array grids of 1-week-old Arabidopsis seedlings as the starting material for imaging. Using this procedure, we screened ~25 000 chemically mutagenised M2 Arabidopsis seeds and recovered photorespiration phenotypes (reduction in Fv/Fm at low CO2) at a frequency of ~4 per 1000 seeds. In addition, we also recovered mutants that showed reduced Fv/Fm at high CO2. Of this group, we detected a novel 'reverse photorespiration' phenotype that showed a high CO2 dependent reduction in Fv/Fm. This chlorophyll fluorescence screening technique promises to reveal novel mutants associated with photorespiration and photoinhibition.

Gene transfer to plants by diverse species of bacteria.

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology. However, the complexity of the patent landscape has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile 'open source' platform for plant biotechnology, may lead to new uses of natural bacteria-plant interactions to achieve plant transformation.