Derek T . A . Lamport (original) (raw)
Papers by Derek T . A . Lamport
Academia Biology, Dec 17, 2024
In The Power of Movement in Plants, Charles Darwin details many examples of oscillatory growth, r... more In The Power of Movement in Plants, Charles Darwin details many examples of oscillatory growth, recently exemplified at the single-cell level by pollen tube tip oscillations and associated ion fluxes, particularly of Ca2+and H+. This implies an underlying growth oscillator, supported by the recent discovery that classical arabinogalactan proteins (AGPs) bind Ca2+ at the cell surface. The juxtaposition of AGPs with three additional components embedded in the plasma membrane provides evidence of a Ca2+ cycle that generates cytosolic Ca2+. This cycle involves Ca2+ channels, auxin efflux “PIN” proteins, and an auxin-activated proton pump that dissociates AGP-Ca2+ on demand. While the apparent simplicity of this system satisfies Occam’s razor, its proposed role as a global growth oscillator demands in-depth examination. The wide ramifications extend from pollen tubes to stomatal guard cells. Stomata act as crucial regulatory components of a water hypercycle that contributes to the homeostasis of a warming planet by regulating evaporative cooling and reflective cloud cover generated by vast tropical rainforests of the South and the equally vast arboreal forests of the North. Finally, forests and the high albedo of snow-capped mountains and polar ice caps are essential to the Gaia hypothesis of James Lovelock and Lynn Margulis, which remains a brilliant metaphor despite earlier criticism.
Cellular and Molecular Life Sciences, Sep 1, 2001
This review of the living cell wall and its protein components is in two parts. The first is anec... more This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.
Experimental Cell Research, 1964
THE application of microbiological techniques to higher plants via the cell suspension technique ... more THE application of microbiological techniques to higher plants via the cell suspension technique is potentially useful in studying almost all aspects of plant metabolism. Thus recent work bears out Haberlandt's [12] prediction that cell suspension cultures would allow the experimental study of many important problems from a new vantage point. For example, the primary cell wall-part instrument, part result of morphogenesis-as the major component of the cell and the ultimate site of auxin action, is uniquely visible from such a vantage point. Cell suspension cultures allow a study of the primary cell wall from the novel consideration that the wall is a "cell particle" possessing structural integrity and a certain degree of enzymic autonomy [17-21]' Or consider the study of organised development in cultured carrot cells. It is reported [41] that" ... cells withdrawri from the phloem of the storage carrot root and which have passed through many transfers in which they were reduced to the single cellular state, have developed into cell aggregates, which have, in turn, differentiated to form roots and, when transplanted, have given rise also to shoots and to a secondarily thickened storage carrot root." These workers do not indicate whether they obtained a single cell clone by the "nurse" tissue technique [28-29] before finally "reconstituting" an entire carrot plant. Despite the usefulness of the cell suspension technique, it is surprising ,1 to note how little the technique has been used for metabolic studies since its introduction by Nickell [31]. The technique's main advantage is the ease with which the fairly large amount of an optically homogeneous, rapidly growing and metabolically active pipeiiable tissue can be grown and used intact or homogenised. The use of sycamore cell suspension cultures, for instance, led to the demonstration of a specific hydroxyproline-rich cell wall protein (provisionally named "extensiu") which may be involved in the control of cell wall extension [17-21]. Some aspects of hydroxyproline bio
Plant Physiology, Jul 1, 1991
Separation of the wound exudate from Acacia senegal (L.) Wildd, "gum arabic," on a preparative Su... more Separation of the wound exudate from Acacia senegal (L.) Wildd, "gum arabic," on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large (-400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small (-30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rodlike macromolecules. The "wattle-blossom" model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall. Gums are of considerable commercial importance and, as products of the specific wound response, gummosis, also of ' Supported by a grant from Pepsico, Inc., and DOE grant No. DE-AC02-76ERO-1 338. 2Dedicated to the memory of Dr. Michael A. Jermyn. 'Abbreviations: HRGP, hydroxyproline-rich glycoprotein; AGP, arabinogalactan-protein; dGAGP, deglycosylated gum arabic glycoprotein; fplc, fast protein liquid chromatography; GAGP, gum arabic glycoprotein; GAP, gum arabic polysaccharide; HF, hydrogen fluoride; Hyp, hydroxyproline; TEM, transmission electron microscopy.
Nature, Dec 1, 1967
ABSTRACT PRIMARY cell walls contain a protein, extensin, which is rich in trans 4-L-hydroxyprolin... more ABSTRACT PRIMARY cell walls contain a protein, extensin, which is rich in trans 4-L-hydroxyproline1–3. I suggested earlier that this protein could cross link wall polysaccharides4; if some of these cross linkages were labile, it would provide a chemical basis for changes in cell wall plasticity which are necessary for extension of plant cells. The recent isolation of several glycopeptides, rich in hydroxyproline from enzymatic digests of tomato cell walls5, confirmed the inference of a covalent carbohydrate–protein linkage in extensin. The chemical composition and properties of these glycopeptides also led me to the preliminary conclusion that attachment of the carbohydrate is by a glycosidic link through the hydroxyl group of hydroxyproline5. This rather unexpected possibility receives additional support from results presented in this communication, describing the isolation of hydroxyproline-O-glycosides from partial alkaline hydrolysates of tomato cell walls. These cell wall preparations account for more than 95 per cent of the hydroxyproline of the cells3. Most of this hydroxyproline (about 70 per cent) can be released as hydroxyproline glycosides. The method is based on the fact that glycosidic linkages are usually stable to those alkaline conditions which lead to rapid hydrolysis of peptide bonds6.
Plant Physiology, Oct 1, 1971
Advances in Botanical Research, 1966
... The Protein Component of Primary Cell Walls. This article is not included in your organizatio... more ... The Protein Component of Primary Cell Walls. This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevier. ... Excerpt. Note: This is a one-page preview only. ...
The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where ... more The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Academia letters, Apr 6, 2022
Plant Physiology, Jun 1, 1992
Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize c... more Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp3 and Ala-Hyp4 repeats that may be related to the classical dicot Ser-Hyp4 extensin motif by the single T-* G (Ser-Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp-Hyp-2 Abbreviations: HRGP, hydroxyproline-rich glycoprotein; AGP,
Plant Physiol., Suppl.; (United States), Apr 1, 1987
Is the cellulose-extensin warp-weft model of primary cell wall organization generally applicable,... more Is the cellulose-extensin warp-weft model of primary cell wall organization generally applicable, or restricted to dicots. Although wall-bound hydroxyproline is usually a quantitative measure of extensin content, it is not definitive at low hyp levels typical of graminaceous monocots. Therefore the authors searched for putative soluble extensins ionically-bound to the cell wall of maize cell suspension cultures. Fractionation of AlClâ eluates on cellex-P, Cellex-E, and Superose-6 gave two HRGPs CEV and CE1 which compositionally resembled extensins rather than the arabinogalactan HRGPs. CEV and CE1 did not react with Yariv's antigen, but did cross-react with antibodies raised against tomato extensin. A tryptic peptide map of CEV gave some major peptides enriched in hydroxyproline and proline residues, indicating peptide periodicity. TEM of the low-angle shadowed protein gave flexuous rodlike molecules of 80 nm contour length. This is the best evidence to date for extensin in monocots.
Phytochemistry, 1986
We raised four sets of rabbit polyclonal antibodies against two highly glycosylated extensin prec... more We raised four sets of rabbit polyclonal antibodies against two highly glycosylated extensin precursors, PI and P2, before and after hydrogen fluoride-deglycosylation. Use of an indirect non-competitive sandwich ELISA technique to determine antibody-antigen cross-reactivities revealed three epitope classes: 1. Glycosylated; 2. Nonglycosylated in the intact glycoprotein; 3. Exposed only after deglycosylation. Thus polyclonals raised against glycosylated Pl or P2 cross-reacted highly (> 50%) with the heterologous glycosylated antigen, i.e. antibody-antigen pairs Pl /P2 and PZ/Pl, but much less with the deglycosylated antigens dP1 and dP2 (< 25 %), implying that the major epitopes are glycosylated; these probably correspond to hydroxyproline oligoarabinosides. The free sugars D-glucose, D-galactose and L-arabinose did not inhibit antibody-antigen binding, in contrast to free hydroxyproline arabinooligosaccharides which did compete at high levels (20-50 mM). Cross-reactivities towards other related macromolecules were low but positive for the following antibody/antigen pairs: Pi/potato lectin, P2/AGP, but negative towards larch arabinogalactan. Polyclonals dP1 and dP2 (raised against the deglycosylated precursors dP1 and dP2) crossreacted significantly with their homologous glycosylated antigen (reactions dPi/Pl and dP2/P2), but only slightly with their heterologous antigen (reactions dPl/P2 and dP2/Pl). These results imply that nonglycosylated epitopes of the glycosylated antigens Pl and P2 differ markedly from one another, and therefore corroborate primary structure information suggesting Val-Lys-Pro-Tyr-His-Pro as the major nonglycosylated epitope of Pl and Val-Tyr-Lys-Tyr-Lys as the major nonglycosylated epitope of P2.
Plant Physiology, Feb 1, 1990
An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criter... more An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the 'diagnostic' pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence [X] or [Y] which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-[X]-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where [X] is [Val-His-Glu/Lys-Tyr-Pro], while in tomato the insertion sequence [Y] = [Val-Lys-Pro-Tyr-His-Pro] and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp-Hyp-Hyp-[Y]-Thr-Hyp-Val-Tyr-Lys. Based on these data we reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks. Purification of Extensin Monomer We separated 70 to 200 mg of crude monomer on a Bio-Rex 70 (100-200 mesh) column (90 x 1.5 cm) eluted (40 mL/h) with a pH and salt gradient: 250 mL of 30 mM (pH 7.6) sodium phosphate buffer and 250 mL of 30 mm (pH 6.1)
Plant Physiology, Nov 1, 1987
Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). ... more Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AIC3) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 pve several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a fial purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threoninehydroxyproline-rich glycoprotein (THRGP) it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated-a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and pve a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin Pl crossreacted significantly with the THRGP, this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot.
Plant Physiology, Feb 1, 1990
Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from... more Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete posttranslational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the 'P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains altemate with high regularity. The THRGP is not in the polyproline-11 conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HFsolvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot. In 1987, we reported (15) the first isolation and partial characterization of a THRGP2 and HRGP from a gramina-'Supported by the U.S. Department of Agriculture grant No. 88-37261-3682 and the U.S. Department of Energy contract No. DE-AC02-76ERO-1 338. 2 Abbreviations: THRGP, threonine-hydroxyproline-rich glycoprotein; HRGP, hydroxyproline-rich glycoprotein; dTHRGP, deglycosylated threonine-hydroxyproline-rich glycoproteins; dw, dry weight; P1, glycosylated tomato extensin type 1; dPI, deglycosylated tomato extensin type P1; P2, glycosylated tomato extensin type 2; dP2, deglycosylated tomato extensin type 2; HHRGP, histidinehydroxyproline-rich glycoprotein; dHHRGP, deglycosylated histidine-hydroxyproline-rich glycoprotein; HF, anhydrous hydrogen fluoride; OPA, orthophthalaldehyde; HFBA, hepta-fluorobutyric acid; AGP, arabinogalactan protein; AP, alkaline phosphatase. CD, circular dichroism; ABTS, 2,2'-Azino-bis(3-ethylbenzthiazinoline-6-sulfonic acid).
New Phytologist, Oct 9, 2017
Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator... more Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates a periplasmic arabinogalactan glycoprotein-calcium (AGP-Ca 2+) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca 2+ oscillations: Hechtian adhesion between the plasma membrane and the cell wall of the growing tip acts as a piconewton force transducer that couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction opens stretch-activated Ca 2+ channels and activates H +-ATPase proton pump efflux that dissociates periplasmic AGP-Ca 2+ resulting in a Ca 2+ influx that activates exocytosis of wall precursors. Thus, a highly simplified pectic primary cell wall regulates its own synthesis by a Hechtian growth oscillator that regulates overall tip growth. By analogy with the three cryptic inscriptions of the classical Rosetta Stone, the Hechtian Hypothesis translates classical AGP function as a Ca 2+ capacitor, pollen tube guide and wall plasticizer into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.
Springer eBooks, 1989
The extensins are hydroxyproline-rich glycoproteins (HRGPs) insolubilized in the cell walls of hi... more The extensins are hydroxyproline-rich glycoproteins (HRGPs) insolubilized in the cell walls of higher plants (Lamport, 1965). They are highly basic and contain about one third protein which is a flexible rod about 80 nm long having repeated glycosylated blocks of the pentapeptide SER-Hyp-Hyp-Hyp (Smith et al., 1986; Van-Hoist and Varner, 1984). The carbohydrate components are arabinoside oligosaccharides O-linked to the hydroxyproline residues (hydroxyproline arabinosides) (Lamport, 1967) and galactosyl-serine (Lamport el al., 1973).
Academia Biology, Dec 17, 2024
In The Power of Movement in Plants, Charles Darwin details many examples of oscillatory growth, r... more In The Power of Movement in Plants, Charles Darwin details many examples of oscillatory growth, recently exemplified at the single-cell level by pollen tube tip oscillations and associated ion fluxes, particularly of Ca2+and H+. This implies an underlying growth oscillator, supported by the recent discovery that classical arabinogalactan proteins (AGPs) bind Ca2+ at the cell surface. The juxtaposition of AGPs with three additional components embedded in the plasma membrane provides evidence of a Ca2+ cycle that generates cytosolic Ca2+. This cycle involves Ca2+ channels, auxin efflux “PIN” proteins, and an auxin-activated proton pump that dissociates AGP-Ca2+ on demand. While the apparent simplicity of this system satisfies Occam’s razor, its proposed role as a global growth oscillator demands in-depth examination. The wide ramifications extend from pollen tubes to stomatal guard cells. Stomata act as crucial regulatory components of a water hypercycle that contributes to the homeostasis of a warming planet by regulating evaporative cooling and reflective cloud cover generated by vast tropical rainforests of the South and the equally vast arboreal forests of the North. Finally, forests and the high albedo of snow-capped mountains and polar ice caps are essential to the Gaia hypothesis of James Lovelock and Lynn Margulis, which remains a brilliant metaphor despite earlier criticism.
Cellular and Molecular Life Sciences, Sep 1, 2001
This review of the living cell wall and its protein components is in two parts. The first is anec... more This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.
Experimental Cell Research, 1964
THE application of microbiological techniques to higher plants via the cell suspension technique ... more THE application of microbiological techniques to higher plants via the cell suspension technique is potentially useful in studying almost all aspects of plant metabolism. Thus recent work bears out Haberlandt's [12] prediction that cell suspension cultures would allow the experimental study of many important problems from a new vantage point. For example, the primary cell wall-part instrument, part result of morphogenesis-as the major component of the cell and the ultimate site of auxin action, is uniquely visible from such a vantage point. Cell suspension cultures allow a study of the primary cell wall from the novel consideration that the wall is a "cell particle" possessing structural integrity and a certain degree of enzymic autonomy [17-21]' Or consider the study of organised development in cultured carrot cells. It is reported [41] that" ... cells withdrawri from the phloem of the storage carrot root and which have passed through many transfers in which they were reduced to the single cellular state, have developed into cell aggregates, which have, in turn, differentiated to form roots and, when transplanted, have given rise also to shoots and to a secondarily thickened storage carrot root." These workers do not indicate whether they obtained a single cell clone by the "nurse" tissue technique [28-29] before finally "reconstituting" an entire carrot plant. Despite the usefulness of the cell suspension technique, it is surprising ,1 to note how little the technique has been used for metabolic studies since its introduction by Nickell [31]. The technique's main advantage is the ease with which the fairly large amount of an optically homogeneous, rapidly growing and metabolically active pipeiiable tissue can be grown and used intact or homogenised. The use of sycamore cell suspension cultures, for instance, led to the demonstration of a specific hydroxyproline-rich cell wall protein (provisionally named "extensiu") which may be involved in the control of cell wall extension [17-21]. Some aspects of hydroxyproline bio
Plant Physiology, Jul 1, 1991
Separation of the wound exudate from Acacia senegal (L.) Wildd, "gum arabic," on a preparative Su... more Separation of the wound exudate from Acacia senegal (L.) Wildd, "gum arabic," on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large (-400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small (-30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rodlike macromolecules. The "wattle-blossom" model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall. Gums are of considerable commercial importance and, as products of the specific wound response, gummosis, also of ' Supported by a grant from Pepsico, Inc., and DOE grant No. DE-AC02-76ERO-1 338. 2Dedicated to the memory of Dr. Michael A. Jermyn. 'Abbreviations: HRGP, hydroxyproline-rich glycoprotein; AGP, arabinogalactan-protein; dGAGP, deglycosylated gum arabic glycoprotein; fplc, fast protein liquid chromatography; GAGP, gum arabic glycoprotein; GAP, gum arabic polysaccharide; HF, hydrogen fluoride; Hyp, hydroxyproline; TEM, transmission electron microscopy.
Nature, Dec 1, 1967
ABSTRACT PRIMARY cell walls contain a protein, extensin, which is rich in trans 4-L-hydroxyprolin... more ABSTRACT PRIMARY cell walls contain a protein, extensin, which is rich in trans 4-L-hydroxyproline1–3. I suggested earlier that this protein could cross link wall polysaccharides4; if some of these cross linkages were labile, it would provide a chemical basis for changes in cell wall plasticity which are necessary for extension of plant cells. The recent isolation of several glycopeptides, rich in hydroxyproline from enzymatic digests of tomato cell walls5, confirmed the inference of a covalent carbohydrate–protein linkage in extensin. The chemical composition and properties of these glycopeptides also led me to the preliminary conclusion that attachment of the carbohydrate is by a glycosidic link through the hydroxyl group of hydroxyproline5. This rather unexpected possibility receives additional support from results presented in this communication, describing the isolation of hydroxyproline-O-glycosides from partial alkaline hydrolysates of tomato cell walls. These cell wall preparations account for more than 95 per cent of the hydroxyproline of the cells3. Most of this hydroxyproline (about 70 per cent) can be released as hydroxyproline glycosides. The method is based on the fact that glycosidic linkages are usually stable to those alkaline conditions which lead to rapid hydrolysis of peptide bonds6.
Plant Physiology, Oct 1, 1971
Advances in Botanical Research, 1966
... The Protein Component of Primary Cell Walls. This article is not included in your organizatio... more ... The Protein Component of Primary Cell Walls. This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevier. ... Excerpt. Note: This is a one-page preview only. ...
The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where ... more The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Academia letters, Apr 6, 2022
Plant Physiology, Jun 1, 1992
Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize c... more Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp3 and Ala-Hyp4 repeats that may be related to the classical dicot Ser-Hyp4 extensin motif by the single T-* G (Ser-Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp-Hyp-2 Abbreviations: HRGP, hydroxyproline-rich glycoprotein; AGP,
Plant Physiol., Suppl.; (United States), Apr 1, 1987
Is the cellulose-extensin warp-weft model of primary cell wall organization generally applicable,... more Is the cellulose-extensin warp-weft model of primary cell wall organization generally applicable, or restricted to dicots. Although wall-bound hydroxyproline is usually a quantitative measure of extensin content, it is not definitive at low hyp levels typical of graminaceous monocots. Therefore the authors searched for putative soluble extensins ionically-bound to the cell wall of maize cell suspension cultures. Fractionation of AlClâ eluates on cellex-P, Cellex-E, and Superose-6 gave two HRGPs CEV and CE1 which compositionally resembled extensins rather than the arabinogalactan HRGPs. CEV and CE1 did not react with Yariv's antigen, but did cross-react with antibodies raised against tomato extensin. A tryptic peptide map of CEV gave some major peptides enriched in hydroxyproline and proline residues, indicating peptide periodicity. TEM of the low-angle shadowed protein gave flexuous rodlike molecules of 80 nm contour length. This is the best evidence to date for extensin in monocots.
Phytochemistry, 1986
We raised four sets of rabbit polyclonal antibodies against two highly glycosylated extensin prec... more We raised four sets of rabbit polyclonal antibodies against two highly glycosylated extensin precursors, PI and P2, before and after hydrogen fluoride-deglycosylation. Use of an indirect non-competitive sandwich ELISA technique to determine antibody-antigen cross-reactivities revealed three epitope classes: 1. Glycosylated; 2. Nonglycosylated in the intact glycoprotein; 3. Exposed only after deglycosylation. Thus polyclonals raised against glycosylated Pl or P2 cross-reacted highly (> 50%) with the heterologous glycosylated antigen, i.e. antibody-antigen pairs Pl /P2 and PZ/Pl, but much less with the deglycosylated antigens dP1 and dP2 (< 25 %), implying that the major epitopes are glycosylated; these probably correspond to hydroxyproline oligoarabinosides. The free sugars D-glucose, D-galactose and L-arabinose did not inhibit antibody-antigen binding, in contrast to free hydroxyproline arabinooligosaccharides which did compete at high levels (20-50 mM). Cross-reactivities towards other related macromolecules were low but positive for the following antibody/antigen pairs: Pi/potato lectin, P2/AGP, but negative towards larch arabinogalactan. Polyclonals dP1 and dP2 (raised against the deglycosylated precursors dP1 and dP2) crossreacted significantly with their homologous glycosylated antigen (reactions dPi/Pl and dP2/P2), but only slightly with their heterologous antigen (reactions dPl/P2 and dP2/Pl). These results imply that nonglycosylated epitopes of the glycosylated antigens Pl and P2 differ markedly from one another, and therefore corroborate primary structure information suggesting Val-Lys-Pro-Tyr-His-Pro as the major nonglycosylated epitope of Pl and Val-Tyr-Lys-Tyr-Lys as the major nonglycosylated epitope of P2.
Plant Physiology, Feb 1, 1990
An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criter... more An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the 'diagnostic' pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence [X] or [Y] which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-[X]-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where [X] is [Val-His-Glu/Lys-Tyr-Pro], while in tomato the insertion sequence [Y] = [Val-Lys-Pro-Tyr-His-Pro] and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp-Hyp-Hyp-[Y]-Thr-Hyp-Val-Tyr-Lys. Based on these data we reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks. Purification of Extensin Monomer We separated 70 to 200 mg of crude monomer on a Bio-Rex 70 (100-200 mesh) column (90 x 1.5 cm) eluted (40 mL/h) with a pH and salt gradient: 250 mL of 30 mM (pH 7.6) sodium phosphate buffer and 250 mL of 30 mm (pH 6.1)
Plant Physiology, Nov 1, 1987
Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). ... more Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AIC3) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 pve several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a fial purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threoninehydroxyproline-rich glycoprotein (THRGP) it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated-a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and pve a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin Pl crossreacted significantly with the THRGP, this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot.
Plant Physiology, Feb 1, 1990
Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from... more Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete posttranslational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the 'P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains altemate with high regularity. The THRGP is not in the polyproline-11 conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HFsolvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot. In 1987, we reported (15) the first isolation and partial characterization of a THRGP2 and HRGP from a gramina-'Supported by the U.S. Department of Agriculture grant No. 88-37261-3682 and the U.S. Department of Energy contract No. DE-AC02-76ERO-1 338. 2 Abbreviations: THRGP, threonine-hydroxyproline-rich glycoprotein; HRGP, hydroxyproline-rich glycoprotein; dTHRGP, deglycosylated threonine-hydroxyproline-rich glycoproteins; dw, dry weight; P1, glycosylated tomato extensin type 1; dPI, deglycosylated tomato extensin type P1; P2, glycosylated tomato extensin type 2; dP2, deglycosylated tomato extensin type 2; HHRGP, histidinehydroxyproline-rich glycoprotein; dHHRGP, deglycosylated histidine-hydroxyproline-rich glycoprotein; HF, anhydrous hydrogen fluoride; OPA, orthophthalaldehyde; HFBA, hepta-fluorobutyric acid; AGP, arabinogalactan protein; AP, alkaline phosphatase. CD, circular dichroism; ABTS, 2,2'-Azino-bis(3-ethylbenzthiazinoline-6-sulfonic acid).
New Phytologist, Oct 9, 2017
Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator... more Occam's Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates a periplasmic arabinogalactan glycoprotein-calcium (AGP-Ca 2+) capacitor with tip-localized AGPs as the source of tip-focussed cytosolic Ca 2+ oscillations: Hechtian adhesion between the plasma membrane and the cell wall of the growing tip acts as a piconewton force transducer that couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction opens stretch-activated Ca 2+ channels and activates H +-ATPase proton pump efflux that dissociates periplasmic AGP-Ca 2+ resulting in a Ca 2+ influx that activates exocytosis of wall precursors. Thus, a highly simplified pectic primary cell wall regulates its own synthesis by a Hechtian growth oscillator that regulates overall tip growth. By analogy with the three cryptic inscriptions of the classical Rosetta Stone, the Hechtian Hypothesis translates classical AGP function as a Ca 2+ capacitor, pollen tube guide and wall plasticizer into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.
Springer eBooks, 1989
The extensins are hydroxyproline-rich glycoproteins (HRGPs) insolubilized in the cell walls of hi... more The extensins are hydroxyproline-rich glycoproteins (HRGPs) insolubilized in the cell walls of higher plants (Lamport, 1965). They are highly basic and contain about one third protein which is a flexible rod about 80 nm long having repeated glycosylated blocks of the pentapeptide SER-Hyp-Hyp-Hyp (Smith et al., 1986; Van-Hoist and Varner, 1984). The carbohydrate components are arabinoside oligosaccharides O-linked to the hydroxyproline residues (hydroxyproline arabinosides) (Lamport, 1967) and galactosyl-serine (Lamport el al., 1973).