Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.

Arabitol dehydrogenase as a selectable marker for rice.

Arabitol dehydrogenase has been adapted for use as a plant selectable marker. Arabitol is a five-carbon sugar alcohol that can be used by E. coli strain C, but not by the laboratory K12 strains. The enzyme converts the non-plant-metabolizable sugar arabitol into xylulose, which is metabolized by plant cells. Rice was transformed with a plant-expression-optimized synthetic gene using Biolistic-mediated transformation. Selection on 2.75% arabitol and 0.25% sucrose yielded a transformation efficiency (9.3%) equal to that obtained with hygromycin (9.2%). Molecular analyses showed that the atlD gene was integrated into the rice genome of selected plants and was inherited in a Mendelian manner. This study indicates that arabitol could serve as an effective means of plant selection.

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root.

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [(3)H]glucosamine and [(35)S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.

Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots.

The block in the electrogenic H(+) efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H(+) efflux is inhibition of K(+) influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H(+) efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N'-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.The results are discussed in terms of the presumed mode of action of fusicoccin on the plasmalemma ATPase. Inhibition of protein synthesis appears to inactivate the proton channel of the ATPase, possibly as the indirect result of disrupted metabolism. Fusicoccin reactivates or bypasses the blocked channel.

Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera).

Four closely related cDNA clones encoding laccase isoenzymes from xylem tissues of yellow-poplar (Ltlacc2.1-4) were identified and sequenced. The inferred yellow-poplar laccase gene products were highly related to one another (79-91% at the amino acid level) and showed significant similarity to other blue copper oxidases, especially with respect to the copper-binding domains. The encoded proteins had N-terminal signal sequences and 17-19 potential N-linked glycosylation sites. The mature proteins were predicted to have molecular masses of ca. 61 kDa (unglycosylated) and high isoelectric points (pI 9.3-9.5). The canonical copper ligands were conserved, with the exception of a Leu residue associated with the axial position of the Type-1 cupric ion. The residue at this position has been proposed to influence the redox potential of Type-1 cupric ions. Northern blot analysis revealed that the yellow-poplar laccase genes are differentially expressed in xylem tissues. The genes were verified as encoding active laccases by heterologous expression in tobacco cells and demonstration of laccase activity in extracts from transformed tobacco cell lines.

Isolation and characterization of three families of auxin down-regulated cDNA clones.

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.

Forest tree biotechnology.

The forest products industry has traditionally viewed trees as merely a raw, and more or less immutable, natural resource. However, unlike such inanimate resources as metallic ores, trees have the potential to be modified genetically, essentially transmuting lead into gold. Increasingly, modern alchemists are applying the tools of biotechnology in efforts to reduce the biological constraints on forest productivity. Several new methodologies being used to address problems in forest biology are described with respect to their potential impact on forest tree improvement. In addition to addressing problems inherent to the current use of trees for production of pulp and paper or solid wood products, genetic manipulation of trees brings with it the potential to create new industries based on the novel characteristics of transgenic trees, e.g. trees containing transgenes to detoxify specific pollutants could be used in the remediation of sites contaminated with hazardous wastes. Efforts to modify trees through biotechnology are in their infancy, and this review seeks to outline the underpinnings of what will undoubtedly be an area of increased emphasis in the next millennium.

Effect of ATPase inhibitors on cell potential and k influx in corn roots.

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Psi) and K(+) ((86)Rb) influx of corn root tissue over a wide range of K(+) activity. N,N'Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K(+) influx and depolarized Psi at low, but not at high, K(+) activity (K degrees ). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K(+) influx over a wide range of K degrees , with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Psi(p)) above 0.2 millimolar K degrees . Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K(+) influx and Psi(p) at low K degrees can be attributed to inhibition of the plasmalemma ATPase.It is concluded that by DCCD treatment, the energy-linked electrogenic system at high K degrees is separated from the energy-linked K(+) influx system at low K degrees . The results are analyzed in terms of electrical analogue models of the membrane. The presence of two, algebraically additive electrogenic components is indicated; one is better modeled as a current source (system I) and one as a voltage source (system II). No K(+) stimulation of system II is required to produce the observed K degrees dependence of Psi(p).

Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus.

It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151-1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean.

Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KDEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24 kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.

Sequence and Expression of a HSP83 from Arabidopsis thaliana.

A full-length cDNA encoding a heat shock protein (hsp) belonging to the 83 to 90 kilodalton hsp family of Arabidopsis thaliana has been isolated and sequenced. Truncated cDNA clones were isolated by nucleic acid hybridization to a truncated soybean HSP83 cDNA probe and a fragment generated from a Drosophila HSP83 gene. A single strand DNA vector/primer based extension procedure was employed to obtain the full-length cDNA. The level of transcripts homologous to this cDNA (AtHS83) is low in 2-week-old Arabidopsis plants but is rapidly enhanced by elevated temperatures. DNA sequence comparison between this cDNA and hsp83-90 sequences from human, yeast and Drosophila reveal amino acid identities of 63 to 69%, typical identities for interspecies comparisons between hsp83 to 90 kilodalton proteins. Genomic DNA blot analysis performed with probes derived from AtHS83 indicate the presence of a HSP83 gene family estimated to be comprised of at least three genes.

Structure and expression of two auxin-inducible genes from Arabidopsis.

Two genes from Arabidopsis thaliana related to the auxin-inducible Aux28 and Aux22 genes of soybean have been isolated. These genes belong to a small multi-gene family and are similar to the soybean Aux gene family in the sequence of the predicted proteins, intron/exon locations, and auxin-enhanced expression of their transcripts. Application of auxin to 8-day old Arabidopsis plants, 4-day old etiolated seedlings, and suspension culture cells all resulted in enhanced Aux transcript levels. Comparison of the promoter sequences from the soybean and Arabidopsis genes yielded no significant sequence conservation; however, three regions of near sequence identity are present between the two Arabidopsis Aux genes.