Elevated Na(+) and pH influence the production and transport of saxitoxin in the cyanobacteria Anabaena circinalis AWQC131C and Cylindrospermopsis raciborskii†T3.

Saxitoxins (STX), neurotoxic alkaloids, fall under the umbrella of paralytic shellfish toxins produced by marine dinoflagellates and freshwater cyanobacteria. The genes responsible for the production of STX have been proposed, but factors that influence their expression and induce toxin efflux remain unclear. Here we characterize the putative STX NorM-like MATE transporters SxtF and SxtM. Complementation of the antibiotic-sensitive strain Escherichia coli KAM32 with these transporters decreased fluoroquinolone sensitivity, indicating that while becoming evolutionary specialized for STX transport these transporters retain relaxed specificity typical of this class. The transcriptional response of STX biosynthesis (sxtA) along with that of the STX transporters (sxtM and sxtF from Cylindrospermopsis raciborskii T3, and sxtM from Anabaena circinalis AWQC131C) were assessed in response to ionic stress. These data, coupled with a measure of toxin intracellular to extracellular ratios, provide an insight into the physiology of STX export. Cylindrospermopsis raciborskii and Anabaena circinalis exhibited opposing responses under conditions of ionic stress. High Na(+) (10 mM) induced moderate alterations of transcription and STX localization, whereas high pH (pH 9) stimulated the greatest physiological response. Saxitoxin production and cellular localization are responsive to ionic strength, indicating a role of this molecule in the maintenance of cellular homeostasis.

Carbon isotope discrimination as a tool to explore C4 photosynthesis.

Photosynthetic carbon isotope discrimination is a non-destructive tool for investigating C4 metabolism. Tuneable diode laser absorption spectroscopy provides new opportunities for making rapid, concurrent measurements of carbon isotope discrimination and CO2 assimilation over a range of environmental conditions, and this has facilitated the use of carbon isotope discrimination as a probe of C4 metabolism. In spite of the significant progress made in recent years, understanding how photosynthetic carbon isotope discrimination measured concurrently with gas exchange relates to carbon isotope composition of leaf and plant dry matter remains a challenge that requires resolution if this technique is to be successfully applied as a screening tool in crop breeding and phylogenetic research. In this review, we update our understanding of the factors and assumptions that underlie variations in photosynthetic carbon isotope discrimination in C4 leaves. Closing the main gaps in our understanding of carbon isotope discrimination during C4 photosynthesis may help advance research aimed at developing higher productivity and efficiency in key C4 food, feed, and biofuel crops.

The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species.

Crop yields need to nearly double over the next 35 years to keep pace with projected population growth. Improving photosynthesis, via a range of genetic engineering strategies, has been identified as a promising target for crop improvement with regard to increased photosynthetic yield and better water-use efficiency (WUE). One approach is based on integrating components of the highly efficient CO(2)-concentrating mechanism (CCM) present in cyanobacteria (blue-green algae) into the chloroplasts of key C(3) crop plants, particularly wheat and rice. Four progressive phases towards engineering components of the cyanobacterial CCM into C(3) species can be envisaged. The first phase (1a), and simplest, is to consider the transplantation of cyanobacterial bicarbonate transporters to C(3) chloroplasts, by host genomic expression and chloroplast targeting, to raise CO(2) levels in the chloroplast and provide a significant improvement in photosynthetic performance. Mathematical modelling indicates that improvements in photosynthesis as high as 28% could be achieved by introducing both of the single-gene, cyanobacterial bicarbonate transporters, known as BicA and SbtA, into C(3) plant chloroplasts. Part of the first phase (1b) includes the more challenging integration of a functional cyanobacterial carboxysome into the chloroplast by chloroplast genome transformation. The later three phases would be progressively more elaborate, taking longer to engineer other functional components of the cyanobacterial CCM into the chloroplast, and targeting photosynthetic and WUE efficiencies typical of C(4) photosynthesis. These later stages would include the addition of NDH-1-type CO(2) pumps and suppression of carbonic anhydrase and C(3) Rubisco in the chloroplast stroma. We include a score card for assessing the success of physiological modifications gained in phase 1a.

Antisense reduction of NADP-malic enzyme in Flaveria bidentis reduces flow of CO2 through the C4 cycle.

An antisense construct targeting the C(4) isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO(2) to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO(2) assimilation rates at high intercellular CO(2) were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO(2) (the ratio of CO(2) leak rate from the bundle sheath to the rate of CO(2) supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C(4) cycle rate, bundle sheath leak rate, and bundle sheath CO(2) concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C(3) and C(4) cycles that exist during environmental perturbations by light and CO(2) can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C(4) pathway could potentially be improved through a reduction in C(4) cycle activity or increased C(3) cycle activity.

Functional analysis of corn husk photosynthesis.

The husk surrounding the ear of corn/maize (Zea mays) has widely spaced veins with a number of interveinal mesophyll (M) cells and has been described as operating a partial C(3) photosynthetic pathway, in contrast to its leaves, which use the C(4) photosynthetic pathway. Here, we characterized photosynthesis in maize husk and leaf by measuring combined gas exchange and carbon isotope discrimination, the oxygen dependence of the CO(2) compensation point, and photosynthetic enzyme activity and localization together with anatomy. The CO(2) assimilation rate in the husk was less than that in the leaves and did not saturate at high CO(2), indicating CO(2) diffusion limitations. However, maximal photosynthetic rates were similar between the leaf and husk when expressed on a chlorophyll basis. The CO(2) compensation points of the husk were high compared with the leaf but did not vary with oxygen concentration. This and the low carbon isotope discrimination measured concurrently with gas exchange in the husk and leaf suggested C(4)-like photosynthesis in the husk. However, both Rubisco activity and the ratio of phosphoenolpyruvate carboxylase to Rubisco activity were reduced in the husk. Immunolocalization studies showed that phosphoenolpyruvate carboxylase is specifically localized in the layer of M cells surrounding the bundle sheath cells, while Rubisco and glycine decarboxylase were enriched in bundle sheath cells but also present in M cells. We conclude that maize husk operates C(4) photosynthesis dispersed around the widely spaced veins (analogous to leaves) in a diffusion-limited manner due to low M surface area exposed to intercellular air space, with the functional role of Rubisco and glycine decarboxylase in distant M yet to be explained.

Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry.

In C(4) plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C(4) dicot Flaveria bidentis grown at a low (LI; 150 micromol quanta m(2) s(-1)) and medium (MI; 500 micromol quanta m(2) s(-1)) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO(2) assimilation per leaf area measured at 1500 micromol quanta m(2) s(-1) were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO(2) (phi) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. phi increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (S(m)) and bundle sheath surface area per unit leaf area (S(b)) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO(2) diffusion were greater in MI compared with LI plants. Despite being a C(4) species, F. bidentis is very plastic with respect to growth irradiance.

Host selection of symbiotic cyanobacteria in 31 species of the Australian cycad genus: Macrozamia (Zamiaceae).

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.