Pea Plumules Research Articles

Biochemical characterization of a family of proteins that stabilizes a plant Ran protein in its GTP-bound conformation

Ran-binding proteins (RanBP) are a group of proteins that bind to Ran (Ras-related nuclear small G-protein) and thus control the GTP/GDP-bound states of the Ran and couple the Ran GTPase cycle to cellular processes. In an effort to identify potential downstream effectors for PsRan1-dependent cellular processes, we detected a group of pea Ran (PsRan1)-binding proteins and characterized their biochemical activities. A Ran overlay assay using [ 32P-GTP]-labeled PsRan1 revealed three PsRan1-binding proteins (33, 45, and 85 kDa in size) from total protein extracts of dark-grown pea plumules. These proteins bound preferentially to the Ran-GTP over Ran-GDP conformation and subsequently stabilized its GTP-bound status. We propose that they are a family of proteins that maintain the Ran protein in the active conformation and are potential downstream mediators for PsRan1-dependent cellular processes. Our report provides the basis for characterizing and dissecting Ran downstream targets and Ran-mediated events, and it thus facilitates our understanding about the roles played by Ran/RanBP signaling pathways during plant growth and development.

Immunological and biochemical evidence for nuclear localization of annexin in peas

Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea ( Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

Characterization of a cytosolic phosphatase from pea plumules having significant protein tyrosine phosphatase activity

An enzyme with phosphatase activity has been purified to near homogeneity from the cytosolic fraction of pea ( Pisum sativum L. cv Alaska) plumules. The enzyme has an estimated molecular mass of between 42 kDa (SDS-PAGE) and 60 kDa (molecular sieve HPLC). It has an isoelectric point range from 6.2 to 6.5. The enzyme was characterized using para-nitrophenyl phosphate ( pNPP) and 32P-labeled proteins as substrates. The enzyme acted as an acid phosphatase (APase, EC 3.1.3.2) with a pH optimum from 4.5 to 6.5 when pNPP was used as substrate. However, its pH optimum shifted to pH 7.0 when 32P-Tyr-poly(Glu,Tyr) was used as substrate, and under these conditions its enzymatic properties were very similar to those of protein tyrosine phosphatases (PTPases, EC 3.1.3.48). The enzyme could be inhibited by micromolar concentrations of molybdate and vanadate, but not by the APase inhibitors citrate and tartrate, or by the protein Ser/Thr phosphatase inhibitors, okadaic acid and microcystin-LR. It did not require Ca 2+, Mg 2+, or Mn 2+ for its activity. In addition to 32P-Tyr-poly(Glu,Tyr), the enzyme could also dephosphorylate 32P-Tyr-myelin basic protein phosphorylated by c-src kinase, but not the P-Tyr-mitogen-activated protein kinase (MAPK) phosphorylated by MAPK kinase. It showed little activity toward 32P-Ser/Thr residues on casein and histone phosphorylated by cAMP-dependent protein kinase (PKA). The sequence of the N-terminal 23 amino acid residues of the phosphatase shows no significant similarity to that of known PTPases from other species, but it had 75 % identity with the N-terminus of a sequence translated from a cDNA of castor bean ( Ricinus communis L.). The overall amino acid sequence of the castor seed polypeptide closely matched that of a purple acid phosphatase isolated from kidney bean ( Phaseolus vulgaris L.).

Red light-induced appearance of phosphotyrosine-like epitopes on nuclear proteins from pea (Pisum sativum L.) plumules.

As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyrosine antibodies in several pea nuclear proteins. The immunostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phytochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immunostained by anti-phosphotyrosine antibodies can be solubilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo.

Partial purification and characterization of a type 1 protein phosphatase in purified nuclei of pea plumules.

We report the isolation and characterization of a protein Ser/Thr phosphatase from highly purified pea nuclei. In subnuclear fractions, more than 75% of Ser/Thr protein phosphatase activity was associated with the chromatin fraction, whereas the other 25% was in the nuclear membrane/nucleoplasmic fraction when phosphorylase a was used as a substrate. The enzyme was purified approx. 2750-fold to a specific activity of approx. 4000 nmol/min per mg. The molecular mass of the enzyme was 34 kDa as estimated by molecular sieve chromatography, and approx. 40 kDa as estimated by SDS/PAGE. The phosphatase was inhibited by okadaic acid with an IC50 of approx. 15 nM, by rabbit muscle inhibitor 2 with an IC50 of approx. 10 nM, and by microcystin-LR with an IC50 of approx. 0.05 nM. The enzyme did not require Ca2+, Mg2+ or Mn2+ for its activity; instead, these cations showed some inhibitory effects. It was inhibited by NaF or citrate but not by tartrate, molybdate or vanadate under the conditions tested. Its sensitivities towards the various phosphatase inhibitors and its substrate specificity were very similar to those characteristic of the type I Ser/Thr protein phosphatases well studied in animal systems. The enzyme was able to selectively dephosphorylate a 92 kDa nuclear protein that had been phosphorylated by one or more endogenous protein kinases.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea.

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

Immunolocalization of an annexin-like protein in corn

Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca 2+ in growth regulation has been discovered. Our recent studies on a Ca 2+-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalization analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro.

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei.

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

Identification of an ARF Type Low Molecular Mass GTP-Binding Protein in Pea (Pisum sativum)

The ARF class of low molecular mass GTP-binding proteins, originally detected as cholera toxin-activated ADP-ribosylation factors (ARF) in mammalian cells, is now known to participate in intracellular membrane vesicle trafficking. We identified a GTP-binding protein in pea plumules which resembles ARF in several respects. Like mammalian ARF, the pea protein had an apparent molecular mass of 21 kDa and was distributed mainly (∼97%) in the cytosol, with 1% and 1.5% occurring in the Golgi and microsomal fractions, respectively. In comparison, small GTP-binding proteins in the 26-30 kDa range were enriched in membranous fractions. The 21 kDa protein crossreacted strongly with an ARF class I antibody prepared against a mammalian ARF, but did not crossreact with ARF 5 (of class II) antibodies. An anti-Volvox yptl antibody did not show immunoreactivity to the 21 kDa pea protein, although it crossreacted strongly with a 28 kDa pea plumule protein. Our results strongly suggest that the 21 kDa pea protein is an analog of the ARF protein localized in the cytosol of mammalian cells. As far as we are aware, this is the first observation of an ARF protein in plants.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei.

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Purification and immunolocalization of an annexin-like protein in pea seedlings

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds (45)Ca(2+) in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei.

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

Spermine stimulation of a nuclear NII kinase from pea plumules and its role in the phosphorylation of a nuclear polypeptide.

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684). In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg2+ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [gamma-32P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.

Nuclear envelope isolation in peas

Nuclear envelopes were isolated from pea plumules by sonication of a highly purified nuclear fraction. The nuclear envelope fraction, examined in the electron microscope by both negative staining and thin sectioning techniques, was found to consists of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents frequently could be recognized. The chemical composition of the nuclear envelope fraction was determined and compared with that of the nuclear fraction. Significant amounts of DNA and RNA were found to remain associated with the nuclear envelope fraction. It was found that the specific activities of ATPase, 5′-nucleotidase and glucose-6-phosphatase were higher in the nuclear envelope fraction than in the total nuclei.

The effect of red light on the flavonoid composition of etiolated pea plumules.

The effect of red light on the flavonoid composition of etiolated pea plumules.