Allan M. Showalter

Structure and function of plant cell wall proteins.

Plant cell walls are amazingly complex amalgams of carbohy? drates, proteins, lignin, water, and incrusting substances such as cutin, suberin, and certain inorganic compounds that vary among plant species, cell types, and even neighboring cells. Developmental events and exposure to any of a number of abiotic and biotic stresses further increase this compositional and structural variation. Moreover, the dynamic nature and func? tions of plant cell walls in terms of growth and development, environmental sensing and signaling, plant defense, intercel? lular communication, and selective exchange interfaces are reflected in these variations. Much is currently known about the structure and metabolic regulation of the various cell wall components, but relatively little is known about their precise functions and intermolecular interactions. In this review, I will discuss the accumulated structural and regulatory data and the much more limited functional and in? termolecular interaction information on five plant cell wall protein classes. These five protein classes, listed in Table 1, include the extensins, the glycine-rich proteins (GRPs), the proline-rich proteins (PRPs), the solanaceous lectins, and the arabinogalactan proteins (AGPs). These five proteins may be evolutionarily related to one another, most obviously because each of them, with the exception of the GRPs, contains hydroxyproline, and less obviously in the case of the GRPs because this class has nucleotide sequence similarity to the extensins. For completeness, I should mention that these are not the only cell wall proteins that are known. Others exist, such as cysteine-rich thionins, 28and 70-kD water-regulated proteins, a histidine-tryptophan-rich protein, and many cell wall enzymes such as peroxidases, phosphatases, invertases, a-mannosidases, P-mannosidases, p-1,3-glucanases, (3-1,4glucanases, polygalacturonase, pectin methylesterases, malate dehydrogenase, arabinosidases, a-galactosidases, (3-galactosidases, |3-glucuronosidases, p-xylosidases, proteases, and ascorbic acid oxidase (Varner and Lin, 1989). However, the above five classes generally represent the most abundant, and to date, the most well-studied and widely documented, plant cell wall proteins. Before describing these five wall protein classes, I should point out that research on these individual proteins has oc? curred in several plant species, but relatively few examples exist where these cell wall proteins have been studied together in one plant, let alone in one particular plant organ or type of cell. Thus, data from one plant species are often extrapolated to represent the situation in other plant species. Although such extrapolations are usually valid, enough variations are now known that caution should be exercised in making or believing such claims.

Arabinogalactan-proteins: structure, expression and function

Abstract. Arabinogalactan-proteins (AGPs) are a family of extensively glycosylated hydroxyproline-rich glycoproteins that are thought to have important roles in various aspects of plant growth and development. After a brief introduction to AGPs highlighting the problems associated with defining and classifying this diverse family of glycoproteins, AGP structure is described in terms of the protein component (including data from molecular cloning), carbohydrate component, processing of AGPs (including recent data on glycosylphosphatidylinositol membrane anchors) and overall molecular shape. Next, the expression of AGPs is examined at several different levels, from the whole plant to the cellular levels, using a variety of experimental techniques and tools. Finally, AGP function is considered. Although the existing functional evidence is not incontrovertible, it does clearly point to roles for AGPs in vegetative, reproductive, and cellular growth and development as well as programmed cell death and social control. In addition and most likely inextricably linked to their functions, AGPs are presumably involved in molecular interactions and cellular signaling at the cell surface. Some likely scenarios are discussed in this context. AGPs also have functions of real or potential commercial value, most notably as emulsifiers in the food industry and as potential immunological regulators for human health. Several important questions remain to be answered with respect to AGPs. Clearly, elucidating the unequivocal functions of particular AGPs and relating these functions to their respective structures and modes of action remain as major challenges in the years ahead.

A Bioinformatics Approach to the Identification, Classification, and Analysis of Hydroxyproline-Rich Glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Hybrid and chimeric versions of HRGP molecules also exist. In order to “mine” genomic databases for HRGPs and to facilitate and guide research in the field, the BIO OHIO software program was developed that identifies and classifies AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs from proteins predicted from DNA sequence data. This bioinformatics program is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs. HRGPs identified by the program are subsequently analyzed to elucidate the following: (1) repeating amino acid sequences, (2) signal peptide and glycosylphosphatidylinositol lipid anchor addition sequences, (3) similar HRGPs via Basic Local Alignment Search Tool, (4) expression patterns of their genes, (5) other HRGPs, glycosyl transferase, prolyl 4-hydroxylase, and peroxidase genes coexpressed with their genes, and (6) gene structure and whether genetic mutants exist in their genes. The program was used to identify and classify 166 HRGPs from Arabidopsis (Arabidopsis thaliana) as follows: 85 AGPs (including classical AGPs, lysine-rich AGPs, arabinogalactan peptides, fasciclin-like AGPs, plastocyanin AGPs, and other chimeric AGPs), 59 EXTs (including SP5 EXTs, SP5/SP4 EXTs, SP4 EXTs, SP4/SP3 EXTs, a SP3 EXT, “short” EXTs, leucine-rich repeat-EXTs, proline-rich extensin-like receptor kinases, and other chimeric EXTs), 18 PRPs (including PRPs and chimeric PRPs), and AGP/EXT hybrid HRGPs.

Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans.

Arabinogalactan-proteins (AGPs) are complex glycoconjugates that are commonly found at the cell surface and in secretions of plants. Their location and diversity of structures have made them attractive targets as modulators of plant development but definitive proof of their direct role(s) in biological processes remains elusive. Here we overview the current state of knowledge on AGPs, identify key challenges impeding progress in the field and propose approaches using modern bioinformatic, (bio)chemical, cell biological, molecular and genetic techniques that could be applied to redress these gaps in our knowledge.

Effects of salinity on growth, ion content, and osmotic relations in Halopyrum mucronatum (L.) Stapf.

Abstract Halopyrum mucronatum (L.) Stapf. is a perennial grass found on the coastal dunes of Karachi, Pakistan. Halopyrum mucronatum plants were grown in 0, 90,180, and 360 mol m‐3 NaCl in a sand culture using a sub‐irrigation method. Fresh and dry weight of roots and shoots peaked at 90 mol m‐3 NaCl. A further increase in salinity inhibited plant growth, ultimately resulting in plant death at 360 mol m‐3 NaCl. The relative growth rate of plants was highest between 60 and 90 days after final salinity concentrations were reached. Maximum succulence was noted in 90 mol m‐3 NaCl. Water potential and osmotic potential of plants became more negative with an increase in salinity, while plants lost turgor with increasing salinity. Time of harvest did not have any significant effect on the water relations of plants. Sodium (Na) and chloride (Cl) content of plants increased with an increase in salinity, while calcium (Ca), magnesium (Mg), and potassium (K) content decreased. Glycinebetaine content of shoots increa...

Effects of sodium chloride treatments on growth and ion accumulation of the halophyte Haloxylon recurvum

Abstract Effects of increasing salt concentrations 0, 180, 360 mol im3 sodium chloride (NaCl), on growth, succulence, mineral composition, and glycinebetaine content in Haloxylon recurvum was investigated. Fresh and dry weight of plants increased with an increase in salinity. Succulence of shoots increased at low salinity and decreased at high salinity. Root and shoot Ca+, Mg+, and K+content decreased with increasing salinity while both Na+ and Cl‐ content increased, reaching 4,900 and 5,300 mmol kg‐1 dry weight, respectively. Glycinebetaine (mol m‐3 tissue water) significantly increased in shoots at 360 mol m‐3 NaCl, but did not differ significantly in roots treated with from 0 to 360 mol m‐3 NaCl. Haloxylon recurvum is a highly salt tolerant stem succulent plant which accumulate a high quantity of salt, which makes it a good candidate to use for phytoremediation in highly saline areas of the sub‐tropics.

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in λgt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2–6-Tyr-Pro and(Gly)2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2–5-Arg repeats.

Effect of salinity on growth, ion content, and cell wall chemistry in Atriplex prostrata (Chenopodiaceae).

Atriplex prostrata Boucher, a facultative halophyte, exhibits significant reduction in height and biomass and in the width of the cortex and vascular tissue under saline conditions. Therefore, the goal of this investigation was to determine the effect of salinity on plant growth as well as on the patterns of lignification, peroxidase activity, and extensin deposition. Biomass, leaf area, internode length, water potential, photosynthesis, transpiration, and ion content were measured. In addition, lignin, peroxidase, and extensin were, respectively, examined via phloroglucinol staining, peroxidase staining, and immunostaining with extensin antibody on tissue prints of free-hand stem sections. Length of internodes and leaf area significantly decreased with increased salinity, and net photosynthesis declined dramatically as well. There was a significant accumulation of Na+ in organs when plants were grown in saline solutions, while the concentration of K+, Ca2+, and Mg2+ decreased. The signals in tissue prints showed that soluble peroxidase and extensin accumulated in the first three internodes of A. prostrata grown under saline conditions. In contrast, lignification was reduced under saline growth conditions in the third and fourth internodes. These results indicate that extensin may replace lignin in providing mechanical support for cells, while stems remain in a juvenile stage because of growth retardation caused by salinity.

Molecular Interactions of Arabinogalactan Proteins with Cortical Microtubules and F-Actin in Bright Yellow-2 Tobacco Cultured Cells

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and β-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with β-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. β-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. β-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.

Cloning and developmental/stress-regulated expression of a gene encoding a tomato arabinogalactan protein

Arabinogalactan proteins (AGPs) represent a major class of plant hydroxyproline-rich glycoproteins (HRGPs) and are components of cell walls and plasma membranes. AGPs are thought to play roles in cell differentiation, development, and cell-cell interactions. Using a synthetic DNA oligonucleotide based upon an amino acid sequence motif common to AGPs from Lolium, rose, and carrot (i.e., Hyp-Ala-Hyp-Ala-Hyp), we have isolated and sequenced the first AGP gene from a partial Sau3A tomato genomic library packaged in bacteriophage charon 35. The deduced 215 amino acid protein contains 20% Ala, 22% Pro, 10% Gly, and 11% Ser and consists of two Pro-Ala-Pro-Ala-Pro pentapeptide repeats and 16 Ala-Pro dipeptide repeats, consistent with known AGP amino acid compositions and sequences. Comparison of the genomic sequence to a reverse transcribed PCR product and tomato cDNA confirmed the AGP gene is expressed and contains one large intervening sequence. RNA blot hybridization analysis in tomato indicates this AGP gene is strongly expressed in stem and flower, moderately expressed in root and green fruit, and weakly expressed in leaves and red fruit as a 980 nucleotide transcript. Five-day-old seedlings also express this transcript; however, this expression is not regulated by light. More significantly, a gradient of AGP gene expression is observed in tomato stems, ranging from high levels of expression in young internodes to low levels of expression in old internodes. Wounding serves to down-regulate expression in young and old internodes. Heat shock also affects AGP gene expression in stems by transiently down-regulating mRNA levels.