Arabidopsis plants expressing only the redox-regulated Rca-α isoform have constrained photosynthesis and plant growth.

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca-α and Rca-ß. Redox modulation of Rca-α regulates the function of Rca-α and Rca-ß acting together to control Rubisco activation. Although Arabidopsis Rca-α alone less effectively activates Rubisco in vitro, it is not known how CO2 assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca-α in the absence of Rca-ß ('Rca-α only' lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca-α only lines, and they maintained a lower rate of CO2 assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca-α only are less effective in initiating and sustaining the activation of Rubisco than when Rca-ß is also present. Currently there are no examples of any plant species that naturally express Rca-α only but numerous examples of species expressing Rca-ß only. That Rca-α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.

The CO2 concentrating mechanism and photosynthetic carbon assimilation in limiting CO2 : how Chlamydomonas works against the gradient.

The CO2 concentrating mechanism (CCM) represents an effective strategy for carbon acquisition that enables microalgae to survive and proliferate when the CO2 concentration limits photosynthesis. The CCM improves photosynthetic performance by raising the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), simultaneously enhancing carbon fixation and suppressing photorespiration. Active inorganic carbon (Ci) uptake, Rubisco sequestration and interconversion between different Ci species catalyzed by carbonic anhydrases (CAs) are key components in the CCM, and an array of molecular regulatory elements is present to facilitate the sensing of CO2 availability, to regulate the expression of the CCM and to coordinate interplay between photosynthetic carbon metabolism and other metabolic processes in response to limiting CO2 conditions. This review intends to integrate our current understanding of the eukaryotic algal CCM and its interaction with carbon assimilation, based largely on Chlamydomonas as a model, and to illustrate how Chlamydomonas acclimates to limiting CO2 conditions and how its CCM is regulated.

Flow rate and duty cycle effects in lysis of Chlamydomonas reinhardtii using high-energy pulsed focused ultrasound.

To consider microalgae lipid biofuel as a viable energy source, it is a necessity to maximize algal cell lysis, lipid harvest, and thus biofuel production versus the energy used to lyse the cells. Previous techniques have been to use energy consumptive ultrasound waves in the 10-40 kHz range in a stationary exposure environment. This study evaluated the potential of using 1.1 MHz ultrasound pulses in a new flow through type chamber on Chlamydomonas reinhardtii as a model organism for cell breakage. The ultrasound was generated using a spherically focused transducer with a focal length of 6.34 cm and an active diameter of 6.36 cm driven by 20 cycle sine-wave tone bursts at varied pulse repetition frequencies. First, variations in flow rate were examined at a constant duty cycle of 3.6%. After assessing flow rates, the duty cycle was varied to further explore the dependence on the tone burst parameters. Cell lysis was assessed by quantifying protein and chlorophyll release into the supernatant as well as by lipid extractability. Appropriate flow rates with higher duty cycles led to statistically significant increases in cell lysis relative to controls and other exposure conditions.

Lysis of Chlamydomonas reinhardtii by high-intensity focused ultrasound as a function of exposure time.

Efficient lysis of microalgae for lipid extraction is an important concern when processing biofuels. Historically, ultrasound frequencies in the range of 10-40 kHz have been utilized for this task. However, greater efficiencies might be achievable if higher frequencies could be used. In our study, we evaluated the potential of using 1.1 MHz ultrasound to lyse microalgae for biofuel production while using Chlamydomonas reinhardtii as a model organism. The ultrasound was generated using a spherically focused transducer with a focal length of 6.34 cm and an active diameter of 6.36 cm driven by 20 cycle sine-wave tone bursts at a pulse repetition frequency of 2 kHz (3.6% duty cycle). The time-average acoustic power output was 26.2 W while the spatial-peak-pulse-average intensity (ISPPA) for each tone burst was 41 kW/cm(2). The peak compressional and rarefactional pressures at the focus were 102 and 17 MPa, respectively. The exposure time was varied for the different cases in the experiments from 5s to 9 min and cell lysis was assessed by quantifying the percentage of protein and chlorophyll release into the supernate as well as the lipid extractability. Free radical generation and lipid oxidation for the different ultrasound exposures were also determined. We found that there was a statistically significant increase in lipid extractability for all of the exposures compared to the control. The longer exposures also completely fragmented the cells releasing almost all of the protein and chlorophyll into the supernate. The cavitation activity did not significantly increase lipid oxidation while there was a minor trend of increased free radical production with increased ultrasound exposure.

Alterations in photosynthesis in Arabidopsis lacking IMMUTANS, a chloroplast terminal oxidase.

Green and white variegation in the Arabidopsis immutans (im) mutant is caused by a nuclear recessive gene. The green sectors contain cells with normal-appearing chloroplasts, while cells in the white sectors have photooxidized plastids lacking organized lamellae. In the present experiments, we found that the green im sectors have enhanced rates of carbon assimilation (monitored by (14)CO(2) uptake) and that there are corresponding increases in the activities of Rubisco and SPS, elevated starch and sucrose pool sizes, and an altered pattern of carbohydrate partitioning that favors sucrose over starch. We hypothesize that these increases are due, at least in part, to interactions with white sectors, perhaps to compensate for reductions in total source tissue. Consistent with this idea, the im white sectors accumulate low levels of sucrose and acid invertase activities are markedly increased in the white versus green cells. This suggests that there is a sucrose gradient between the green and white sectors, and that sucrose is transported from the green to white cells in response to sink demand. The expression of photosynthetic genes is not appreciably altered in the green im sectors versus wild type, but rather there is an up-regulation of genes involved in defense against oxidative stress and down-regulation of genes involved in cell wall biosynthesis. We postulate that changes in photosynthesis in the im green cells are driven by a need for photoprotection (especially early in chloroplast biogenesis) and due to source-sink interactions.