Focus Issue on Plant Cell Walls: Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.Purple acid phosphatase (PAP) belongs to a large family of dinuclear metalloenzymes (LeBansky et al., 1992; Klabunde et al., 1996) and catalyzes the hydrolysis of a wide range of phosphate esters. It is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron (III) charge transfer transition (Antanaitis et al., 1983). Arabidopsis (Arabidopsis thaliana) contains a large family of PAPs composed of 29 genes, 28 of which have signal peptides that potentially transfer to the wall and/or vacuole. Only a few functions have been suggested for these phosphatases: AtPAP15 seems to modulate ascorbic acid biosynthesis (Zhang et al., 2008), and AtPAP17 may play a role in the metabolism of reactive oxygen species (del Pozo et al., 1999). In other plant species, soybean (Glycine max) GmPAP3 is induced by NaCl stress but not by phosphorus deficiency (Liao et al., 2003), tomato (Solanum lycopersicum) PAP may release phosphate from extracellular phosphate ester under phosphate starvation (Bozzo et al., 2002), and tobacco (Nicotiana tabacum) NtPAP12 could be involved in the deposition of β-glucan (Kaida et al., 2003, 2009; Sano et al., 2003). Mammalian PAPs, which are secretory enzymes, may be involved in iron transport (Nuttleman and Roberts, 1990), generation of reactive oxygen species (Sibille et al., 1987), and bone resorption (Ek-Rylander et al., 1994).We previously demonstrated that the activities of cellulose and callose synthases are enhanced by overexpression of NtPAP12 in tobacco cells (Kaida et al., 2009). The phosphorylation/dephosphorylation process in those synthases may occur directly on the catalytic subunit itself, which has been predicted to be located on the cytoplasmic side of the plasma membrane (Nühse et al., 2004; Taylor, 2007). This is not compatible with the cell wall localization of NtPAP12. The data also indicate that phosphorylation may play a role in regulating the turnover of cellulose synthase by proteolysis through a proteasome-dependent pathway (Taylor, 2007), which again implies a cytoplasmic phosphorylation event. Thus, we suggested that NtPAP12 could be involved in the regulation of cellulose synthase activity, either by acting on an unidentified membrane protein or by enhancing its activity with an effector, which can lead to the promotion of cellulose synthesis. Nevertheless, this phosphatase may be involved in the activation of synthases indirectly by acting on either apoplastic proteins or unidentified membrane proteins, since the level of activation for glucan synthases was only a 2- to 3-fold increase in the transgenic tobacco cells overexpressing NtPAP12 compared with wild-type cells.The extracellular phosphorylation network has been proposed by proteomic analysis of Arabidopsis cells due to the identification of phosphorylated Tyr residues in xyloglucanase, putative lectin receptor-like kinase, and putative chitinase (Ndimba et al., 2003). The change in phosphorylation status was also identified in the extracellular peroxidase in maize (Zea mays) cells (Chivasa et al., 2005b). Another analysis has indicated that some potential phosphorylated proteins might be present in the apoplastic space during wall regeneration (Kwon et al., 2005). We previously showed that tobacco PAP had a higher catalytic efficiency for Tyr phosphopeptides (k_cat/K_m = 1,093–1,335) than for ATP (k_cat/K_m = 333) and p-nitrophenyl-phosphate (k_cat/K_m = 379), suggesting that the enzyme could dephosphorylate the phosphoryl residues of proteins in vivo (Kaida et al., 2008). There is still much to be learned, however, including the role that phosphorylation plays in the functions of these proteins. It is possible, for example, that extracellular PAPs might modify the functions of the phosphoproteins by dephosphorylating those proteins in the apoplasts, but to date no evidence has been reported demonstrating this activity. In this study, we searched for substrates of PAP using phosphoproteomic analyses of apoplastic proteins in tobacco cells.     

Cellulose and Callose Biosynthesis in Higher Plants (I. Solubilization and Separation of (1->3)- and (1->4)-[beta]-Glucan Synthase Activities from Mung Bean)

(1->3)- and (1->4)-[beta]-glucan synthase activities from higher plants have been physically separated by gel electrophoresis in nondenaturing conditions. The two glucan synthases show different mobilities in native polyacrylamide gels. Further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a different polypeptide composition in these synthases. Three polypeptides (64, 54, and 32 kD) seem to be common to both synthase activities, whereas two polypeptides (78 and 38 kD) are associated only with callose synthase activity. Twelve polypeptides (170, 136, 108, 96, 83, 72, 66, 60, 52, 48, 42, and 34 kD) appear to be specifically associated with cellulose synthase activity. The successful separation of (1->3)- and (1->-4)-[beta]-glucan synthase activities was based on the manipulation of digitonin concentrations used in the solubilization of membrane proteins. At low dipitomin concentrations (0.05 and 0.1%), the ratio of the cellulose to callose synthase activity was higher. At higher digitonin (0.5-1%) concentrations, the ratio of the callose to cellulose synthase activity was higher. Rosette-like particles with attached product were observed in samples taken from the top of the stacking gel, where only cellulose was synthesized. Smaller (nonrosette) particles were found in the running gel, where only callose was synthesized. These findings suggest that a higher level of subunit organization is required for in vitro cellulose synthesis in comparison with callose assembly.     

Callose synthesis in higher plants

Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches and it exists in the cell walls of a wide variety of higher plants. Callose plays important roles during a variety of processes in plant development and/or in response to multiple biotic and abiotic stresses. It is now generally believed that callose is produced by callose synthases and that it is degraded by β-1,3-glucanases. Despite the importance of callose in plants, we have only recently begun to elucidate the molecular mechanism of its synthesis. Molecular and genetic studies in Arabidopsis have identified a set of genes that are involved in the biosynthesis and degradation of callose. In this mini-review, we highlight recent progress in understanding callose biosynthesis and degradation and discuss the future challenges of unraveling the mechanism(s) by which callose synthase operate.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, pollen, plasmodesmata, cell plate, stress     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Callose (β-1,3 glucan) is essential for <Emphasis Type="Italic">Arabidopsis</Emphasis>pollen wall patterning,but not tube growth

Background  

Callose (β-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis β-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Interaction between Cytokinesis-Related Callose and Cortical Microtubules in Dividing Cells of the Liverwort Riella helicophylla

Abstract: In juvenile walls of dividing cells of the liverwort Riella helicophylla the nitroso-derivative of photolysed Nifedipine (a calcium antagonist) stimulates the deposition of callose. This enhanced biosynthesis of β-1,3-glucan can only be observed in the cell plate, the juvenile cell walls and the walls of adjacent cells. An immunocytological analysis of this effect revealed that no cortical microtubules occurred at the sites of callose deposition. The cells of the control displayed a normal distribution of cortical microtubules at the plasma membrane as long as no callose was deposited along the corresponding walls. In a second set of experiments, inhibitors of microtubule polymerization and depolymerization (amiprophosmethyl and taxol, respectively) were used. At low concentrations, these substances also caused a significant stimulation of callose deposition in the plane of cell division. Based on these findings, we propose a regulatory model of callose and cellulose biosynthesis that depends on the binding of the cellulose/callose synthase complex to cortical microtubules that may be mediated by unknown binding protein(s).     

Plant callose synthase complexes

Synthesis of callose (-1,3-glucan) in plants has been a topic of much debate over the past several decades. Callose synthase could not be purified to homogeneity and most partially purified cellulose synthase preparations yielded -1,3-glucan in vitro, leading to the interpretation that cellulose synthase might be able to synthesize callose. While a rapid progress has been made on the genes involved in cellulose synthesis in the past five years, identification of genes for callose synthases has proven difficult because cognate genes had not been identified in other organisms. An Arabidopsis gene encoding a putative cell plate-specific callose synthase catalytic subunit (CalS1) was recently cloned. CalS1 shares high sequence homology with the well-characterized yeast -1,3-glucan synthase and transgenic plant cells over-expressing CalS1 display higher callose synthase activity and accumulate more callose. The callose synthase complex exists in at least two distinct forms in different tissues and interacts with phragmoplastin, UDP-glucose transferase, Rop1 and, possibly, annexin. There are 12 CalS isozymes in Arabidopsis, and each may be tissue-specific and/or regulated under different physiological conditions responding to biotic and abiotic stresses.     

Role of Enzymes in Growth and Morphology of Neurospora crassa: Cell-Wall-Bound Enzymes and Their Possible Role in Branching

The cell wall of Neurospora crassa contains bound enzymes that can digest its structural polymers. These enzymes are not present at the same levels at all stages of growth. The levels of these autolytic enzymes vary and generally show some relationship to the process of branching. These enzymes were removed from the cell wall by β-mercaptoethanol extraction and were tested for activity against isolated cell wall fractions. Such studies, as well as autolytic studies, showed that enzymes acting on the protein portion of the cell wall (proteases) are more prominent than enzymes that act on the glucan portion (glucanases) of the cell wall. Comparative studies between the wild type and a spreading colonial mutant spco-1 showed that earlier and higher frequency of branching in spco-1 was correlated with a greater amount of these enzymes bound to the cell walls. It is concluded from these observations that autolytic enzymes acting on the protein and glucan portion of the cell walls occur as wall-bound and participate in the process of branching in Neurospora.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.     

A cell plate-specific callose synthase and its interaction with phragmoplastin

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-beta-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate--associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate--specific enzyme is demonstrated by the observations that the green fluorescent protein--CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.     

Regulation of Cell Wall Synthesis by the Clathrin Light Chain Is Essential for Viability in Schizosaccharomyces pombe

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.     

Biosynthesis of non-cellulosic polysaccharides of plant cell walls

Enzymes that make the polymer backbones of plant cell wall polysaccharides have proven to be recalcitrant to biochemical purification. Availability of mutational genetics and genomic tools paved the way for rapid progress in identifying genes encoding various cell wall glycan synthases. Mutational genetics, the primary tool used in unraveling cellulose biosynthesis, was ineffective in assigning function to any of the hemicellulosic, polymerizing glycan synthases. A combination of comparative genomics and functional expression in a heterologous system allowed identification of various cellulose synthase-like (Csl) sequences as being involved in the formation of β-1,4-mannan, β-1,4-glucan, and mixed-linked glucan. A number of xylose-deficient mutants have led to a variety of genes, none of which thus far possesses the motifs known to be conserved among polymerizing β-glycan synthases. Except for xylan synthase, which appears to be an agglomerate of proteins just like cellulose synthase, Golgi glycan synthases already identified suggest that the catalytic polypeptide by itself is sufficient for enzyme activity, most likely as a homodimer. Several of the Csl genes remain to be assigned a function. The possibility of the involvement of various Csl genes in making more than one product remains.     

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.