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DRGs are highly conserved GTP binding proteins. All eukaryotes examined contain DRG1 and DRG2 orthologs. The first experimental evidence for GTP binding by a plant DRG1 protein and by DRG2 from any organism is presented. DRG1 antibodies recognized a single ∼43‐kDa band in plant tissues, whereas DRG2 antibodies recognized ∼45‐, 43‐, and 30‐kDa bands. An in vitro transcription and translation assay suggested that the 45‐kDa band represents full‐length DRG2 and that the smaller bands are specific proteolytic products. Homogenates from pea roots and root apices were used to produce fractions enriched in cytosolic and microsomal monosomes and polysomes. DRG1 and the 45‐ and 43‐kDa DRG2 bands occurred in the cytosol and associated with cytosolic monosomes. In contrast, the 30‐kDa form of DRG2 was strongly enriched in polysome fractions. Thus, DRG1 and the larger forms of DRG2 may be involved in translational initiation, and the 30‐kDa form of DRG2 may be involved in translational elongation. DRG1 and the 45‐ and 43‐kDa forms of DRG2 can reassociate with ribosomes in vitro, a process that is partially inhibited by GTP‐γ‐S. Cells expressing FLAG‐tagged ribosomal proteins from transgenic lines ofArabidopsisand yeast also demonstrated DRG‐ribosome interactions.

DRGs are very highly conserved GTP-binding proteins. All eukaryotes contain DRG1 and DRG2 orthologs. Arabidopsis has three DRGs: AtDRG1 (At4g39520), AtDRG2 (At1g17470), and AtDRG3 (At1g72660). DRG2 and DRG3 encode proteins that are 95% identical; identity between DRG1 and DRG2/3 is 55%. The focus of this article is expression of Arabidopsis DRGs. DRG1 and DRG2 promoter-GUS constructs showed similar spatial expression in seedlings and mature organs, but gene-specific differences were noted. Quantitative real-time PCR experiments indicated similar levels of DRG1 and DRG2 mRNA accumulation in most tissues. DRG3 transcripts were very low in all tissues. Heat stress at 37°C led to a 10-fold increase in DRG1 transcripts and a 1000-fold increase in DRG3 transcripts. DRG1 antibodies recognized a 43-kD protein, and DRG2 antibodies recognized bands at 30, 43, and 45 kD. Plants were exposed to stresses (salt, heat, cold, UV light, osmotic, and other stresses) and examined by Western blotting. Only heat stress caused detectable changes. Heat did not affect DRG1, but DRG2 and a 72-kD protein recognized by DRG2 antibodies both increased. The modest changes in DRG mRNA and protein levels seen here suggest that other types of regulation, such as altered subcellular localization, may be important for their cellular functions.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs. PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots >>root apices; fully-elongated stems > elongating stems), and thus is an excellent \"dormancy\" marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs.

Monomers of the plant cell wall glycoprotein extensin are secreted into the wall where they become cross-linked to each other to form a rigid matrix. Expression of the extensin matrix is correlated with the inhibition of further cell elongation during normal development, with increased resistance to virulent pathogens and with other physiological responses characterized by wall strengthening. Carbohydrates make up about two-thirds of the mass of extensin. Arabinose oligomers linked to hydroxyproline residues represent 95% of the total carbohydrate with the remainder occurring as single residues of galactose linked to some serine residues. Electron microscopy of shadowed extensin shows the glycosylated form to be an easily visualized and highly elongated molecule. In contrast, extensin that has been deglycosylated with anhydrous hydrogen fluoride is difficult to resolve in the EM. Glycosylated extensin elutes from a gel filtration column much more rapidly than does the deglycosylated form, and from this analysis we have calculated respective Stokes' radii of 89 and 11 Ȧngstroms for these molecules. Others have shown that inhibition of extensin glycosylation has no effect on its secretion or insolubilization in the cell wall, but that this extensin cannot inhibit cell elongation. It is likely that carbohydrate moieties keep extensin in an extended conformation and that extensin must be in this conformation to form a cross-linked matrix that can function properly in vivo.

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations greater than or equal to 10 micromolar, bud growth and expression of pGB8 in the buds are inhibited

DRGs are very highly conserved GTP‐binding proteins. All eukaryotes containDRG1andDRG2orthologs.Arabidopsishas threeDRGs:AtDRG1(At4g39520),AtDRG2(At1g17470), andAtDRG3(At1g72660).DRG2andDRG3encode proteins that are 95% identical; identity betweenDRG1andDRG2/3is 55%. The focus of this article is expression ofArabidopsis DRGs.DRG1andDRG2promoter‐GUS constructs showed similar spatial expression in seedlings and mature organs, but gene‐specific differences were noted. Quantitative real‐time PCR experiments indicated similar levels ofDRG1andDRG2mRNA accumulation in most tissues.DRG3transcripts were very low in all tissues. Heat stress at 37°C led to a 10‐fold increase inDRG1transcripts and a 1000‐fold increase inDRG3transcripts.DRG1antibodies recognized a 43‐kD protein, andDRG2antibodies recognized bands at 30, 43, and 45 kD. Plants were exposed to stresses (salt, heat, cold, UV light, osmotic, and other stresses) and examined by Western blotting. Only heat stress caused detectable changes. Heat did not affectDRG1, butDRG2and a 72‐kD protein recognized byDRG2antibodies both increased. The modest changes inDRGmRNA and protein levels seen here suggest that other types of regulation, such as altered subcellular localization, may be important for their cellular functions.

Developmentally regulated GTP-binding proteins (DRGs) from animals and fungi are highly conserved but have no known function. Here we characterize DRGs from pea (PsDRG) and Arabidopsis (AtDRG). Amino acid sequences of AtDRG and PsDRG were 90% identical to each other and about 65% identical to human DRG. Genomic Southern blotting indicated that AtDRG and PsDRG probably are single-copy genes. PsDRG mRNA accumulated preferentially in growing organs (root apices, growing axillary buds and elongating stems) compared with their non-growing counterparts. At DRG mRNA was relatively abundant in Arabidopsis leaves, stems and siliques, less abundant in flowers and flower buds, and barely detectable in roots. Histone mRNAs are known to accumulate predominantly during S phase of the cell cycle and are markers for proliferating cells. The patterns of histone H2A mRNA accumulation in pea and Arabidopsis organs were very similar to those of DRG mRNAs. An antiserum raised against a PsDRG N-terminal fusion protein recognized 43 and 45 kDa proteins. PsDRG proteins were more abundant in growing pea roots and stems than in non-growing organs, but they were equally abundant in growing and dormant axillary buds. After differential centrifugation, PsDRG proteins were found primarily in the microsomal (150,000 x g pellet) and soluble (150,000 x g supernatant) cell fractions.

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as \"extensin-1\" to distinguish it from \"extensin-2,\" a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated (≅84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots ≫root apices; fully-elongated stems >elongating stems), and thus is an excellent \"dormancy\" marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs.

PsSat5, a cDNA clone fromPisum sativumcv. Alaska, contained a microsatellite consisting of 15 TCA repeats within the 5′UTR. This SSR microsatellite was immediately upstream of the presumptive ATG start codon. PCR amplification of genomic DNA from cv. Alaska yielded an identical sequence. This repeat region was analyzed from 10 additional wild and cultivated accessions ofPisumand from four closely related genera (Cicer,Lathyrus,Lens, andVicia). All of the sequences were generally quite similar, with the exception of the number of TCA repeats (region 3) and a short domain immediately upstream of the repeats (region 2).Pisum humile‐northern andLathyruseach contained four TCA repeats (the fewest number observed). Similar toP. sativum‐Alaska and other cultivated peas,Lenscontained 15 repeats, the largest number observed. The number of TCA repeats does not appear to correspond to the established phylogeny of these accessions, so the cellular events that generated variable numbers of repeats probably have occurred repeatedly and have involved both expansions and contractions in the number of repeats. The mRNA corresponding to PsSat5 was found in all tissues ofP. sativum‐Alaska that were examined, but its abundance in leaves and sepals was low. The level of expression was similar in growing and nongrowing stems, roots, and axillary buds. Northern blot analysis of stems and leaves of all 15 accessions showed similar levels of expression. Therefore, there is not a clear correlation between the number of TCA repeats in the 5′UTR and the level of Sat5 expression.