A novel combination of plant growth regulators for in vitro regeneration of complete plantlets of guar [Cyamopsis tetragonoloba (L.) Taub].

A novel combination of plant growth regulators comprising indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and gibberellic acid (GA3) in Murashige and Skoog basal medium has been formulated for in vitro induction of both shoot and root in one culture using cotyledonary node explants of guar, (Cyamopsis tetragonoloba). Highest percentages of shoot (92%) and root (80%) induction were obtained in the medium containing (mg/L) 2 IBA, 3 BA and 1 GA3. Shoot regeneration from the cotyledonary node explants was observed after 10-15 days. Regeneration of roots from these shoots occurred after 20 to 25 days. The regenerated plantlets showed successful acclimatization on transfer to soil. This protocol is expected to be helpful in carrying out various in vitro manipulations in this economically and industrially important legume.

Building the wall: genes and enzyme complexes for polysaccharide synthases.

The complete sequence of the Arabidopsis genome has revealed a total of 40 cellulose synthase (CesA) and cellulose synthase-like (Csl) genes. Recent studies suggest that each CESA polypeptide contains only one catalytic center, and that two or more polypeptides from different genes might be needed to form a functional cellulose synthase complex.

A 55 kDa plasma membrane-associated polypeptide is involved in beta-1,3-glucan synthase activity in pea tissue.

By glycerol gradient centrifugation of a detergent-solubilized plasma membrane fraction from pea tissue, we find a polypeptide of 55 kDa that copurifies with beta-1,3-glucan synthase activity. An antiserum against this polypeptide adsorbs glucan synthase activity and the 55 kDa polypeptide from digitonin-solubilized plasma membrane. These results indicate that the 55 kDa polypeptide is involved in pea beta-1,3-glucan synthase activity.

Isoelectric Focusing of Plant Plasma Membrane Proteins : Further Evidence that a 55 Kilodalton Polypeptide Is Associated with beta-1,3-Glucan Synthase Activity from Pea.

Detergent-solubilized plasma membrane proteins from pea (Pisum sativum L.) stem tissue were separated by isoelectric focusing (IEF) using a Bio-Rad Rotofor cell, with the goal of identifying protein(s) involved in beta-1,3-glucan synthase (GS-II) activity. Ordinary IEF procedures result in membrane protein precipitation. Inclusion of 10% glycerol mitigates this problem in digitonin-solubilized preparations, but not in those solubilized in 3-[(-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Loss of GS-II activity during IEF is minimized by improved cooling of the Rotofor cell. GS-II focuses at pH 5.1. Antiserum against a 55 kilodalton (kD) polypeptide that was recognized from other evidence as involved in GS-II activity, detects this polypeptide in exact correspondence with the GS-II activity peak. A presumptive P-type ATPase, detected using an antibody against corn root plasma membrane 97 kD ATPase, focuses at pH 5.3. In this digitonin/glycerol medium, most of the membrane proteins focus within the relatively narrow pH range of 4.5 to 6, compared to pH 5.5 to 8.5 for IEF in the presence of 9 molar urea, 2% Nonidet P-40 (NP-40), and 5% mercaptoethanol, a medium that inactivates GS-II. This latter medium increases the apparent isoelectric point (pl) values of the abovementioned 55 and 97 kD polypeptides to 5.8 and 7.3, respectively. In the digitonin/glycerol medium, membrane polypeptides apparently focus at pH values lower than their true pls, because of adhering negatively charged phospholipids, which can be at least partially removed by the detergent NP-40 in the presence of urea. These results provide independent evidence that the 55 kD polypeptide is associated with the GS-II activity and indicate that inclusion of urea and a strong nonionic detergent such as NP-40 is necessary if membrane proteins are to be focused at pH values near their true pls.

Purification of 1,3-beta-D-glucan synthase activity from pea tissue. Two polypeptides of 55 kDa and 70 kDa copurify with enzyme activity.

From pea plasma membranes isolated by aqueous polymer two-phase partitioning we have purified 1,3-beta-D-glucan synthase [glucan synthase-II (GS-II) or callose synthase], an enzyme that several reports have suggested consists of between six and nine different subunits. The procedure involves (a) preliminary removal of peripheral proteins by 0.1% digitonin; (b) solubilization of GS-II with 0.5% digitonin; (c) precipitation of activity-irrelevant proteins from the digitonin extract by Ca2+, spermine and cellobiose, which are GS-II effectors needed in step (d); (d) product entrapment by formation of 1,3-beta-D-glucan from UDP-Glc by GS-II in the presence of the mentioned effectors, followed by centrifugal sedimentation of product micelles and elution of proteins therefrom with buffer; (e) preparative isoelectric focusing (IEF) of product-entrapped proteins; and (f) glycerol gradient centrifugation of the fractions of peak GS-II activity from IEF. The procedure yields 300-fold enrichment of GS-II specific activity over that in isolated plasma membranes, and 5500-fold over that in the original homogenate. Out of approximately six principal polypeptides that occur after the product entrapment step, the glycerol gradient GS-II activity peak contains only two major polypeptides, one of 55 kDa and another of 70 kDa, plus minor amounts of one or two others whose distribution and occurrence indicate are not responsible for GS-II activity. Antisera against either the 55-kDa or the 70-kDa polypeptide adsorb more than 60% of the GS-II activity from a product-entrapped preparation. After native gel electrophoresis, GS-II activity is associated with a single protein band of very large molecular mass, whose principal components are the 55-kDa and 70-kDa polypeptides, accompanied by minor amounts of a few other polypeptides most of which do not occur in enzyme preparations purified by the previously described procedure. The 55-kDa but not the 70-kDa component can be labeled by ultraviolet irradiation of the plasma membranes in the presence of [alpha-32P]UDP-Glc under GS-II assay conditions. It seems likely, therefore, that the 55-kDa and 70-kDa polypeptides form a large catalytic complex of which the 55-kDa component is the UDP-Glc-binding subunit.

Nitrate absorption by corn roots : inhibition by phenylglyoxal.

Nitrate transport in excised corn (Zea mays L.) roots was inhibited by phenylglyoxal, but not by 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) or fluorescein isothiocyanate (FITC). Inhibition of nitrate uptake by a 1-hour treatment with 1 millimolar phenylglyoxal was reversed after 3 hours, which was similar to the time needed for induction of nitrate uptake. If induction of nitrate uptake occurs by de novo synthesis of a nitrate carrier, then the resumption of nitrate uptake in the inhibitor-treated roots may occur because of turnover of phenylglyoxal-inactivated nitrate carrier proteins. All three chemicals inhibited chloride uptake to varying degrees, with FITC being the strongest inhibitor. While inhibition due to DIDS was reversible within 30 minutes, both FITC and phenylglyoxal showed continued inhibition of chloride uptake for up to 3 hours after removal from the uptake solution. Assuming that the anion transporter polypeptide(s) carries a positive charge density at or near the transport site, the results indicate that the nitrate carrier does not carry any lysyl residues that are accessible to DIDS or FITC, whereas the chloride carrier does. Both chloride and nitrate carriers, however, seem to possess arginyl residues that are accessible to phenylglyoxal.

Correlated induction of nitrate uptake and membrane polypeptides in corn roots.

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the (35)S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: purification, gene cloning, and trans-Golgi localization.

We purified from pea (Pisum sativum) tissue an approximately 40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Plant polypeptides reversibly glycosylated by UDP-glucose. Possible components of Golgi beta-glucan synthase in pea cells.

In pea membranes, UDP[14C]Glc glycosylates a approximately 40-kDa polypeptide doublet. This label rapidly disappears if excess unlabeled UDP-Glc, or UDP, is added, indicating that the glycosylation is reversible, and suggesting that the glycosylated polypeptides might be intermediates in a glycosyl transfer reaction. Glycosylation of the doublet requires a divalent cation, the effective ions being the same (except for Zn2+) as those that activate Golgi-localized beta-glucan synthase (GS-I) activity. Treatments that inhibit GS-I also inhibit doublet glycosylation. The doublet is associated with Golgi (and to a minor extent with plasma) membranes and occurs also in the soluble fraction. The Golgi-bound doublet may be a component of the GS-I system. Immunological, inactivation, and fractionation evidence indicates that at least one other polypeptide is required in GS-I activity.

A comparative analysis of the plant cellulose synthase (CesA) gene family.

CesA genes are believed to encode the catalytic subunit of cellulose synthase. Identification of nine distinct CesA cDNAs from maize (Zea mays) has allowed us to initiate comparative studies with homologs from Arabidopsis and other plant species. Mapping studies show that closely related CesA genes are not clustered but are found at different chromosomal locations in both Arabidopsis and maize. Furthermore, sequence comparisons among the CesA-deduced proteins show that these cluster in groups wherein orthologs are often more similar than paralogs, indicating that different subclasses evolved prior to the divergence of the monocot and dicot lineages. Studies using reverse transcriptase polymerase chain reaction with gene-specific primers for six of the nine maize genes indicate that all genes are expressed to at least some level in all of the organs examined. However, when expression patterns for a few selected genes from maize and Arabidopsis were analyzed in more detail, they were found to be expressed in unique cell types engaged in either primary or secondary wall synthesis. These studies also indicate that amino acid sequence comparisons, at least in some cases, may have value for prediction of such patterns of gene expression. Such analyses begin to provide insights useful for future genetic engineering of cellulose deposition, in that identification of close orthologs across species may prove useful for prediction of patterns of gene expression and may also aid in prediction of mutant combinations that may be necessary to generate severe phenotypes.