C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.     

Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant.

Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.     

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.     

A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins

The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.     

Targeting of two Arabidopsis H(+)-ATPase isoforms to the plasma membrane.

More than 11 different P-type H(+)-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H(+)-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1-193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H(+)-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H(+)-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.     

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.     

The plant plasma membrane H(+)-ATPase: structure, function and regulation

The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport. In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms. At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes. Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species. In the near future, we can expect regulatory aspects of gene expression to be elucidated. Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties. It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region. Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity. The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information. Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated. In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context.     

Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H+ -ATPase that is functional in yeast and developmentally regulated

PnPMA1, a gene encoding a putative P-type plasma membrane H(+)-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1,068 amino acid residues and a molecular mass of 116.3kDa. PnPMA1 shows moderate identity (30-50%) to plant and fungal plasma membrane H(+)-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H(+)-ATPases but also has a distinct domain of approximately 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.     

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.     

Summary

The plasma membrane H⁺-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H⁺-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H⁺-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14–3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14–3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14–3-3 protein binding motif in Raf-1. The results demonstrate that the 14–3-3 protein is an activator molecule of the H⁺-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H⁺-ATPase.     

The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform

The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.     

Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure-function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H(+)-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.     

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.     

Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM H+ -ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM H+ -ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+ -ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+ -ATPase regulation.     

Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum.

Recombinant plant plasma membrane H(+)-ATPase has been produced in a yeast expression system comprising a multicopy plasmid and the strong promoter of the yeast PMA1 gene. Western blotting with a specific monoclonal antibody showed that the plant ATPase is one of the major membrane proteins made by the transformed cells, accounting for about 1% of total yeast protein. The plant ATPase synthesized in yeast is fully active. It hydrolyzes ATP, pumps protons, and the reaction cycle involves a phosphorylated intermediate. Phosphorylation is possible from both ATP and Pi. Unlike the situation in plants, however, most of the plant ATPase is not expressed in the yeast plasma membrane. Rather, the enzyme appears to remain trapped at a very early stage of secretory pathway: insertion into the endoplasmic reticulum. This organelle was observed to proliferate in the form of stacked membranes surrounding the yeast nucleus in order to accommodate the large amount of plant ATPase produced. In this location, the plant ATPase can be purified with high yield (70 mg from 1 kg of yeast) from membranes devoid of endogenous yeast plasma membrane H(+)-ATPase. This convenient expression system could be useful for other eukaryotic membrane proteins and ATPases.     

Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein

The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.     

Evidence for a plasma membrane proton pump in phloem cells of higher plants

Metabolic energy is required for the loading of sucrose into the phloem and translocation of sugars throughout the plant. The proton electrochemical gradient generated by a plasma membrane proton pump (H(+)-ATPase) is thought to provide energy for these processes. The plasma membrane H(+)-ATPase is encoded by a multigene family in Arabidopsis thaliana. Here we characterize the expression of isoform AHA3 (Arabidopsis H(+)-ATPase isoform 3). The AHA3 mRNA start site was mapped and 464 bp of the putative upstream regulatory region sequenced. A translational fusion of AHA3 to the beta-glucuronidase (GUS) reporter gene was constructed and used to generate transgenic Nicotiana and Arabidopsis plants. Using a histochemical stain, expression of the AHA3/GUS fusion was found predominantly in phloem cells of leaves, stems, roots, and flowers. Biochemical measurements of GUS activity in pith and vascular explants confirmed the histochemical localization. Our results support the hypothesis that a proton pump is present in phloem cells, possibly providing energy to drive plasma membrane cotransport systems required for phloem loading and translocation of photosynthates. In addition to AHA3/GUS expression in phloem, expression was observed in pollen and regions of the ovule, tissues whose physiological functions correlate with a requirement for high levels of solute transport.