Jon K. Pittman

The potential of sustainable algal biofuel production using wastewater resources

The potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production.

Protein phylogenetic analysis of Ca2+/cation antiporters and insights into their evolution in plants

Cation transport is a critical process in all organisms and is essential for mineral nutrition, ion stress tolerance, and signal transduction. Transporters that are members of the Ca2+/cation antiporter (CaCA) superfamily are involved in the transport of Ca2+ and/or other cations using the counter exchange of another ion such as H+ or Na+. The CaCA superfamily has been previously divided into five transporter families: the YRBG, Na+/Ca2+ exchanger (NCX), Na+/Ca2+, K+ exchanger (NCKX), H+/cation exchanger (CAX), and cation/Ca2+ exchanger (CCX) families, which include the well-characterized NCX and CAX transporters. To examine the evolution of CaCA transporters within higher plants and the green plant lineage, CaCA genes were identified from the genomes of sequenced flowering plants, a bryophyte, lycophyte, and freshwater and marine algae, and compared with those from non-plant species. We found evidence of the expansion and increased diversity of flowering plant genes within the CAX and CCX families. Genes related to the NCX family are present in land plant though they encode distinct MHX homologs which probably have an altered transport function. In contrast, the NCX and NCKX genes which are absent in land plants have been retained in many species of algae, especially the marine algae, indicating that these organisms may share “animal-like” characteristics of Ca2+ homeostasis and signaling. A group of genes encoding novel CAX-like proteins containing an EF-hand domain were identified from plants and selected algae but appeared to be lacking in any other species. Lack of functional data for most of the CaCA proteins make it impossible to reliably predict substrate specificity and function for many of the groups or individual proteins. The abundance and diversity of CaCA genes throughout all branches of life indicates the importance of this class of cation transporter, and that many transporters with novel functions are waiting to be discovered.

Emerging mechanisms for heavy metal transport in plants.

Heavy metal ions such as Cu(2+), Zn(2+), Mn(2+), Fe(2+), Ni(2+) and Co(2+) are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd(2+), Hg(2+) and Pb(2+), can become extremely toxic. Thus mechanisms must exist to satisfy the requirements of cellular metabolism but also to protect cells from toxic effects. The mechanisms deployed in the acquisition of essential heavy metal micronutrients have not been clearly defined although a number of genes have now been identified which encode potential transporters. This review concentrates on three classes of membrane transporters that have been implicated in the transport of heavy metals in a variety of organisms and could serve such a role in plants: the heavy metal (CPx-type) ATPases, the natural resistance-associated macrophage protein (Nramp) family and members of the cation diffusion facilitator (CDF) family. We aim to give an overview of the main features of these transporters in plants in terms of structure, function and regulation drawing on information from studies in a wide variety of organisms.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

The Monosaccharide Transporter Gene, AtSTP4, and the Cell-Wall Invertase, Atβfruct1, Are Induced in Arabidopsis during Infection with the Fungal Biotroph Erysiphe cichoracearum

Powdery mildew fungi are biotrophic pathogens that form a complex interface, the haustorium, between the host plant and the parasite. The pathogen acts as an additional sink, competing with host sinks, resulting in considerable modification of photoassimilate production and partitioning within the host tissue. Here, we examine the factors that may contribute to these changes. We show for the first time in one biotrophic interaction (Arabidopsis/Erysiphe cichoracearum) all of the following responses: Glc uptake in host tissues is enhanced after fungal infection; this coincides with the induction of expression of the monosaccharide transporter gene, Arabidopsis sugar transport protein 4 (AtSTP4), in infected leaves; invertase activity and transcript levels for a cell wall invertase, Atβfruct1, increase substantially in Arabidopsis during attack by this pathogen. Before infection, Arabidopsis plants transformed with an AtSTP4 promoter-β-glucuronidase construct show expression mainly in sink tissues such as roots; after infection, AtSTP4 expression is induced in the mature leaves and increases over the 6-d time period. Sections of infected leaves stained for β-glucuronidase show that AtSTP4 expression is not confined to infected epidermal cells but is also evident in a wider range of cells, including those of the vascular tissue. The results are discussed in relation to the possible coordinated expression of hexose transporters and cell wall invertase in the host response to powdery mildew infection.

The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance.

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca2+ sensor, SOS3, have been demonstrated to modulate the plasma membrane H+/Na+ antiporter SOS1; however, how these regulators modulate Ca2+ levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H+/Ca2+ antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H+/Ca2+-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca2+ and Na+ homeostasis in plants.

Functional Association of Arabidopsis CAX1 and CAX3 Is Required for Normal Growth and Ion Homeostasis

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolar H+/Ca2+ transporter, and the closely related transporter CAX3. We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H+/Ca2+ transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca2+ hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca2+ and also displayed a 22% reduction in vacuolar H+-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca2+ and other ions. These growth defects were partially suppressed by addition of exogenous Mg2+. The double mutant displayed a 42% decrease in vacuolar H+/Ca2+ transport, and a 47% decrease in H+-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO43−, Mn2+, and Zn2+ and decreased Ca2+ and Mg2+ in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.

Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters.

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H+/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H+/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.

ECA3, a Golgi-Localized P2A-Type ATPase, Plays a Crucial Role in Manganese Nutrition in Arabidopsis

Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P2A-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Δpmc1 Δpmr1 Δcnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the growth defect of K616 on low Ca. Promoter:β-glucuronidase studies show that ECA3 is expressed in a range of tissues and cells, including primary root tips, root vascular tissue, hydathodes, and guard cells. When transiently expressed in Nicotiana tabacum, an ECA3-yellow fluorescent protein fusion protein showed overlapping expression with the Golgi protein GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly functioning in the transport of these ions into the Golgi. ECA3 is the first P-type ATPase to be identified in plants that is required under Mn-deficient conditions.