Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells

Arabinogalactan proteins (AGPs) are very large proteoglycans thought to have more of a signaling than a structural role when secreted into the plant cell wall. AGPs are also the first known family of abundant plant proteins synthesized with glycosylphosphatidylinositol(GPI) anchors. Nascent cellular Arabidopsis AGPs, still bearing an intact GPI anchor, and AGPs copiously discharged into the culture medium after phospholipase-cleavage of their anchor were each represented by more than 15 seemingly homologous molecular species of increasing size. In washed cells ³H-ethanolamine was slowly incorporated into each AGP’s GPI anchor via phosphatidylethanolamine. Pulse labeling of AGPs by ³H-acetate and by ³H-galactose was much more rapid, allowing labeled AGP detection in the growth medium within 1 h. HPLC analysis of the radiolabel distribution in AGPs secreted within 1–8 h revealed a sharp preference for the larger molecular species. After several hours a population of smaller radioactive AGP species began to appear in the medium. Following certain manipulations of the cells newly secreted AGP species measured by HPLC on a relative mass basis formed a pattern surprisingly different from the radioactivity pattern, although larger species still dominated. Thus Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling.     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives

 The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours. Received: 12 June 1999 / Accepted: 20 October 1999     

Characterization of an arabinogalactan-protein from suspension culture of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Suspension cultures of Echinacea purpurea have been established in MS medium supplemented with 2,4-D and an arabinogalactan-protein (AGP) was purified from the secreted soluble polymers by precipitation with ethanol, followed by precipitation with β-glucosyl Yariv reagent. It revealed typical features of AGPs: a high amount of polysaccharide (90% w/w) with the dominating monosaccharides galactose and arabinose and some glucuronic acid, and a small protein moiety (10% w/w) with the main amino acids Ala, Hyp, Glx, Ser, Asx and Thr. Linkage- and NMR-analyses showed the polysaccharide part to be composed of a branched core-polysaccharide of 3-, 6- and 3,6-linked Galp residues with terminal Araf, Arap, Galp and GlcAp residues. Compared to an AGP from pressed juice of the aerial parts of Echinacea purpurea, differences particularly in terminal arabinose mono- and oligosaccharides in arabinogalactan (AG) side branches could be detected. Testing of different AGP-antibodies with both AGPs confirmed the results of the analytical investigations. Binding of AGPs from plant and cell cultures to LM2, a monoclonal AGP-antibody reacting with a GlcA containing epitope, was comparable. The reactivity of a monoclonal antibody raised against the AGP from the plant recognizing a galactan epitope was also nearly similar with both AGPs. In contrast, polyclonal antibodies raised against the AGP from the plant and directed against an Araf-containing epitope of the AG side branches showed nearly no cross reactivity with the AGP from cell culture.     

NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection inNicotiana alata

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.     

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of microfilaments

The cortical microfilament (MF) component of the cytoskeleton within axial elements of the secondary vascular system of the angiosperm tree, Aesculus hippocastanum L. (horse-chestnut) was studied using transmission electron microscopy of ultrathin sections and indirect immunofluorescence microscopy of actin in thick sections. As seen by electron microscopy, MF bundles have a net axial orientation within fusiform cambial cells and their secondary vascular derivatives (i.e. in the axial xylem and phloem parenchyma, xylem fibres, vessel and sieve elements, and companion cells). Immunofluorescence studies, however, reveal that this axial orientation can be more accurately described as a helix of extremely high pitch; it is a persistent feature of all axial secondary vascular elements during their development. Helical MF arrays are the only arrangement seen in secondary phloem cells. However, in addition to helices, other MF arrays are seen in secondary xylem cells. For example, fibres possess ellipses of MFs associated with simple-pit formation, and vessel elements possess circular arrays of MFs that associate with the developing inter-vessel bordered pits, ray–vessel contact pits, and with the perforation plate. Linear MF arrays are seen co-oriented with the developing tertiary wall-thickenings in vessel elements. The possible roles of MFs during the cytodifferentiation of secondary vascular cells is discussed, and compared with that of microtubules. Received: 7 June 1999 / Accepted: 23 December 1999     

Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis

The exodermal layers that are formed in maize roots during aeroponic culture were investigated with respect to the radial transport of cis-abscisic acid (ABA). The decrease in root hydraulic conductivity (Lp_r) of aeroponically grown roots was stimulated 1.5-fold by ABA (500 nM), reaching Lp_r values of roots lacking an exodermis. Similar to water, the radial flow of ABA through roots (J_ABA) and ABA uptake into root tissue were reduced by a factor of about three as a result of the existence of an exodermis. Thus, due to the cooperation between water and solute transport the development of the ABA signal in the xylem was not affected. This resulted in unchanged reflection coeffcients for roots grown hydroponically and aeroponically. Despite the well-accepted barrier properties of exodermal layers, it is concluded that the endodermis was the more effective filter for ABA. Owing to concentration polarisation effects, ABA may accumulate in front of the endodermal layer, a process which, for both roots possessing and lacking an exodermis, would tend to increase solvent drag and hence ABA movement into the xylem sap at increased water flow (J_Vr). This may account for the higher ABA concentrations found in the xylem at greater pressure difference. Received: 26 January 1999 / Accepted: 26 May 1999     

Identification of a signal peptide for oryzacystatin-I

 A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Received: 29 July 1999 / Accepted: 25 August 1999     

Conversion of guaiacyl to syringyl moieties on the cinnamyl alcohol pathway during the biosynthesis of lignin in angiosperms

Aglycons derived from 4-O-β-D-glucosides of both caffeyl and 5-hydroxyconiferyl alcohols were incorporated into guaiacyl (G) and syringyl (S) units in the lignin of newly formed xylem of several angiosperms. It is likely that these aglycons enter the cinnamyl alcohol pathway as intermediates in the introduction of methoxyl groups onto aromatic rings, and serve as precursors for the biosynthesis of lignin. The S/G ratio in this pathway was coincident with the ratio in the cell wall lignin of each tree. Our results indicate that the cinnamyl alcohol pathway involves the same mechanisms as the cinnamic acid and cinnamyl CoA pathways and they suggest that this novel pathway might be part of a metabolic grid in the biosynthesis of lignin. Received: 8 September 1999 / Accepted: 4 October 1999     

Arabino-Galactan Proteins from <Emphasis Type="Italic">Pistacia lentiscus</Emphasis> var. <Emphasis Type="Italic">chia</Emphasis>: isolation,characterization and biological function

Summary. Arabino-Galactan Proteins (AGPs) were isolated from Chios mastic gum (CMG) by using a buffer containing 0.1 M NaCl, 20 mM Tris–HCl, pH 7.5. Protein analytical methods, combined with specific procedures for carbohydrate characterization, indicated the presence of highly glycosylated protein backbone. In particular, staining by Yariv reagent of the electrophoretically separated molecules revealed the existence of arabinose and galactose and such a modification is characteristic for AGPs. After experiments involving extensive dialysis of the isolated extracts against water and atomic absorption, there was evidence of the existence of zinc ions that are probably covalently bound to the AGPs. By using anion-exchange chromatography, capillary electrophoresis, colorimetric methods and GC-MS, it was found that the extracts were separated into three major populations (A, B, and C), which were consistent with their respective negative charge content namely, uronic acid. The characterization of neutral sugars that was investigated with GC-MS showed the existence of arabinose and galactose in different amounts for each group. Experiments concerning the inhibition of growth of Helicobacter pylori in the presence of AGPs, as is shown for other CMG constituents, showed that the extracts of at least 1.4 g CMG affected the viability of the bacterium. There is no evidence as to whether the AGPs provoke abnormal morphologies of H. pylori, as is reported for the total CMG, or for O-glycans that possess terminal α1, 4-linked N-acetylglucosamine and are expressed in the human gastric mucosa; this has to be further investigated. Authors’ address: Theodora Choli-Papadopoulou, Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, TK 54124 Thessaloniki, Greece     

Different pH-dependences of K+ channel activity in bundle sheath and mesophyll cells of maize leaves

The isolation of bundle sheath protoplasts from leaves of Zea mays L. for patch clamp whole-cell experiments presents special problems caused by the suberin layer surrounding these cells. These problems were overcome by the isolation technique described here. Two different types of whole-cell response were found: a small response caused by MB-1 (maize bundle sheath conductance type 1) which was instantaneously activated, and another caused by MB-2 (maize bundle sheath conductance type 2) consisting of an instantaneous response (maize bundle sheath K⁺ instantaneous current type 2; MB-KI2) similar to but stronger than the current through MB-1 plus a small time-dependent outward rectifying component (maize bundle sheath activated outward rectifying current; MB-AOR) with voltage-dependent delayed activation. The occurrence of MB-AOR was often accompanied by a smaller contribution from an inward rectifying channel at negative potentials. Activation of MB-2 required ATP. It is suggested that MB-1 and MB-2 are related to bundle sheath cells with and without direct contact with the xylem vessels. In mesophyll cells, only one type of response caused by MM-2 (maize mesophyll conductance type 2) was found with an instantaneous (maize mesophyll K⁺ instantaneous current type 2, MM-KI2) and a voltage-dependent delayed component (maize mesophyll activated outward rectifying current, MM-AOR). The most striking difference between bundle sheath and mesophyll cells was the pH dependence of K⁺ uptake. At pH 7.2, uptake of K⁺ by MB-2 was identical to that by MM-2 over the whole voltage range. However, acidification stimulated K⁺ conductance in bundle sheath cells, whereas a decrease was found for MM-2. At pH 6.15, the bundle sheath channel MB-2 had more than a 10-fold higher K⁺ uptake at positive and negative potentials than MM-2. The channel MB-1, too, was stimulated by low pH. This seems to indicate a putative role for MB-1 and MB-2 in charge balance during uptake of nutrients via cotransport from the xylem into the symplasm. Received: 23 April 1999 / Accepted: 19 July 1999     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes

 The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L. Received: 19 July 1999 / Accepted: 19 February 2000     

The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana

 Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N-terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis. Received: 26 August 1999 / Accepted: 4 October 1999     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999