Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum.

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.

Impacts of CO2 Enrichment on Productivity and Light Requirements of Eelgrass.

Seagrasses, although well adapted for submerged existence, are CO2-limited and photosynthetically inefficient in seawater. This leads to high light requirements for growth and survival and makes seagrasses vulnerable to light limitation. We explored the long-term impact of increased CO2 availability on light requirements, productivity, and C allocation in eelgrass (Zostera marina L.). Enrichment of seawater CO2 increased photosynthesis 3-fold, but had no long-term impact on respiration. By tripling the rate of light-saturated photosynthesis, CO2 enrichment reduced the daily period of irradiance-saturated photosynthesis (Hsat) that is required for the maintenance of positive whole-plant C balance from 7 to 2.7 h, allowing plants maintained under 4 h of Hsat to perform like plants growing in unenriched seawater with 12 h of Hsat. Eelgrass grown under 4 h of Hsat without added CO2 consumed internal C reserves as photosynthesis rates and chlorophyll levels dropped. Growth ceased after 30 d. Leaf photosynthesis, respiration, chlorophyll, and sucrose-phosphate synthase activity of CO2-enriched plants showed no acclimation to prolonged enrichment. Thus, the CO2-stimulated improvement in photosynthesis reduced light requirements in the long term, suggesting that globally increasing CO2 may enhance seagrass survival in eutrophic coastal waters, where populations have been devastated by algal proliferation and reduced water-column light transparency.

Carbon Partitioning in Eelgrass (Regulation by Photosynthesis and the Response to Daily Light-Dark Cycles).

Diel variations in rates of C export, sucrose-phosphate synthase (SPS) and sucrose synthase (SS) activity, and C reserves were investigated in Zostera marina L. (eelgrass) to elucidate the environmental regulation of sucrose formation and partitioning in this ecologically important species. Rates of C flux and SPS activity increased with leaf age, consistent with the ontogenic transition from sink to source status. Rates of C export and photosynthesis were low but quantitatively consistent with those of many terrestrial plant species. The Vmax activity of SPS approached that of maize, but substrate-limited rates were 20 to 25% of Vmax, indicating a large pool of inactive SPS. SPS was unresponsive to the day/night transition or to a 3-fold increase in photosynthesis generated by high [CO2] and showed little sensitivity to inorganic phosphate. Consequently, regulation of eelgrass SPS appeared similar to starch- rather than to sugar-accumulating species even though eelgrass accumulates sucrose. Leaf [sucrose] was constant and high throughout the diel cycle, which may contribute to the down-regulation of SPS. Root sucrose synthase activity was high but showed no response to nocturnal anoxia. Root [sucrose] also showed no diel cycle. The temporal stability of [sucrose] confers an ability for eelgrass to buffer the effects of prolonged light limitation that may be key to its survival and ecological success in environments subject to periods of extreme light limitation and chaotic daily variation in light availability.