Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Adaptive signals in algal Rubisco reveal a history of ancient atmospheric carbon dioxide

Rubisco, the most abundant enzyme on the Earth and responsible for all photosynthetic carbon fixation, is often thought of as a highly conserved and sluggish enzyme. Yet, different algal Rubiscos demonstrate a range of kinetic properties hinting at a history of evolution and adaptation. Here, we show that algal Rubisco has indeed evolved adaptively during ancient and distinct geological periods. Using DNA sequences of extant marine algae of the red and Chromista lineage, we define positive selection within the large subunit of Rubisco, encoded by rbcL, to occur basal to the radiation of modern marine groups. This signal of positive selection appears to be responding to changing intracellular concentrations of carbon dioxide (CO(2)) triggered by physiological adaptations to declining atmospheric CO(2). Within the ecologically important Haptophyta (including coccolithophores) and Bacillariophyta (diatoms), positive selection occurred consistently during periods of falling Phanerozoic CO(2) and suggests emergence of carbon-concentrating mechanisms. During the Proterozoic, a strong signal of positive selection after secondary endosymbiosis occurs at the origin of the Chromista lineage (approx. 1.1 Ga), with further positive selection events until 0.41 Ga, implying a significant and continuous decrease in atmospheric CO(2) encompassing the Cryogenian Snowball Earth events. We surmise that positive selection in Rubisco has been caused by declines in atmospheric CO(2) and hence acts as a proxy for ancient atmospheric CO(2).     

The role of phytoplankton photosynthesis in global biogeochemical cycles

Phytoplankton biomass in the world's oceans amounts to only 1–2% of the total global plant carbon, yet these organisms fix between 30 and 50 billion metric tons of carbon annually, which is about 40% of the total. On geological time scales there is profound evidence of the importance of phytoplankton photosynthesis in biogeochemical cycles. It is generally assumed that present phytoplankton productivity is in a quasi steady-state (on the time scale of decades). However, in a global context, the stability of oceanic photosynthetic processes is dependent on the physical circulation of the upper ocean and is therefore strongly influenced by the atmosphere. The net flux of atmospheric radiation is critical to determining the depth of the upper mixed layer and the vertical fluxes of nutrients. These latter two parameters are keys to determining the intensity, and spatial and temporal distributions of phytoplankton blooms. Atmospheric radiation budgets are not in steady-state. Driven largely by anthropogenic activities in the 20th century, increased levels of IR- absorbing gases such as CO₂, CH₄ and CFC's and NO_x will potentially increase atmospheric temperatures on a global scale. The atmospheric radiation budget can affect phytoplankton photosynthesis directly and indirectly. Increased temperature differences between the continents and oceans have been implicated in higher wind stresses at the ocean margins. Increased wind speeds can lead to higher nutrient fluxes. Throughout most of the central oceans, nitrate concentrations are sub-micromolar and there is strong evidence that the quantum efficiency of Photosystem II is impaired by nutrient stress. Higher nutrient fluxes would lead to both an increase in phytoplankton biomass and higher biomass-specific rates of carbon fixation. However, in the center of the ocean gyres, increased radiative heating could reduce the vertical flux of nutrients to the euphotic zone, and hence lead to a reduction in phytoplankton carbon fixation. Increased desertification in terrestrial ecosystems can lead to increased aeolean loadings of essential micronutrients, such as iron. An increased flux of aeolean micronutrients could fertilize nutrient-replete areas of the open ocean with limiting trace elements, thereby stimulating photosynthetic rates. The factors which limit phytoplankton biomass and photosynthesis are discussed and examined with regard to potential changes in the Earth climate system which can lead the oceans away from steady-state. While it is difficult to confidently deduce changes in either phytoplankton biomass or photosynthetic rates on decadal time scales, time-series analysis of ocean transparency data suggest long-term trends have occurred in the North Pacific Ocean in the 20th century. However, calculations of net carbon uptake by the oceans resulting from phytoplankton photosynthesis suggest that without a supply of nutrients external to the ocean, carbon fixation in the open ocean is not presently a significant sink for excess atmospheric CO₂.The submitted paper has been authored under Contract No. DE-AC02-76H00016 with the US Department of Energy. Accordingly, the US Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for US Government purposes.     

An external delta-carbonic anhydrase in a free-living marine dinoflagellate may circumvent diffusion-limited carbon acquisition

The oceans globally constitute an important sink for carbon dioxide (CO(2)) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO(2) and bicarbonate (HCO(3)(-)) is pH dependent, and, in normal, slightly alkaline seawater, [CO(2)] is typically low (approximately 10 mum). Second, the rate of CO(2) diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO(2) from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with delta-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with (125)I. The fixation of (14)CO(2) is strongly pH dependent, suggesting preferential uptake of CO(2) rather than HCO(3)(-), and photosynthetic rates decrease in the presence of 1 mm acetazolamide, a non-membrane-permeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO(2) availability at the cell surface.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms.

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.