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The Physiology and Molecular Biology of Plant 1,3- -D-Glucanases and 1,3;1,4- -D-Glucanases
Abstract
This review covers the physiology and molecular biology of the plant beta-glucanases possessing either endo-1,3-beta-D-giucanase (EC 3.2.1.39) or endo-1,3;1,4-beta-D-glucanase (EC 3.2.1.73) activity. These beta-glucanases are structurally related enzymes that are believed to be involved in many important aspects of plant physiology and development, such as germination, growth, defense against pathogens, flowering, cellular and tissue development and differentiation, and probably other roles. They also are regulated by numerous plant hormones, biotic and abiotic elicitors and stresses, and they exhibit complex tissue- and developmental-specific gene expression.
... Although the major interest in ß-1,3-glucanases stems from their possible role in the response of plants to microbial pathogens, there is strong evidence that these enzymes are also implicated in diverse physiological and developmental processes in the uninfected plant including cell division, 8,9 microsporogenesis, 10,11 pollen germination and tube growth, 12,13 fertilization, 14,15 embryogenesis, 16,16a fruit ripening, 17 seed germination, 18,19 mobilization of storage reserves in the endosperm of cereal grains, 20 bud dormancy, 21 and responses to wounding, cold, ozone and UV B. [22][23][24][25][26] In this chapter we focus on progress being made in understanding the function and regulation of ß-1,3-glucanases in reproductive development and pathogenesis. For more general information on plant ß-1,3-glucanases, the reader is referred to the extensive reviews by Stone and Clarke, 7 Meins et al., 4 Simmons, 6 and Høj and Fincher. 5 ...
... 43 The class II tobacco isoforms are at least 82 % identical in amino acid sequence and differ from the class I enzymes at a minimum of 48.8 % of the positions. 4,6,27,44 Class II also includes the two acidic 41 kDa stylar ßGlu isoforms, Sp41a and Sp41b, which are exclusively expressed in the style of tobacco flowers. 15 They do not appear to be induced by pathogen infection and, hence, are referred to as "PR-like proteins". ...
Several classes of proteins, called pathogenesis-related (PR) proteins, are induced in response to the infection of plants with microbial pathogens. This chapter deals with the family of PR-2 proteins, which are β-1,3-glucanases (glucan endo-1,3-β-glucosidases, E.C. 3.2.1.39) able to catalyze endo-type hydrolytic cleavage of the 1,3-β-D-glucosidic linkages in β-1,3-glucans. The β-1,3-glucanases are abundant, highly regulated enzymes widely distributed in seed-plant species.
... Our observation that Al toxicity induces a l,3-|3-glucanase is consistent with previous reports that these enzymes are induced by other metal ions including Co, Ba, Mn, Hg, and Ag (Fink et al., 1990). Generally, l,3-/3-glucanases play an important role in defense against fungal attack in both dicotyledonous and monocotyledonous plants (Simmons, 1994); however, their role in metal toxicity is not known. Considering that Al toxicity may render plant root cells more leaky (Zhao et al., 1987;Ownby, 1993), we suggest that during Al stress root cells are more susceptible to pathogen attack. ...
... The wheat l,3-j3-glucanase described here is unique, having characteristics of several dissimilar glucanases previously described. For example, it is relatively acidic (with a pi of 6.0), which is the hallmark of glucanases generally secreted to the intercellular spaces (Simmons, 1994). It has a putative N-terminal 25-amino acid signal peptide that is likely to mediate transport into the ER secretory pathway, leading to extracellular secretion. ...
A cDNA library made from mRNA of Al-treated roots of an Al-sensitive wheat (Triticum aestivum cv Victory) cultivar was screened with a degenerate oligonucleotide probe derived from the partial amino acid sequence of the Al-induced protein TAI-18. Of seven clones that initially hybridized with the probe, one encoded a novel 1,3-[beta]-glucanase having a calculated molecular weight of 46.3 and an isoelectric point of 6.0. Like the A6 1,3-[beta]-glucanase gene products from Brassica napus and Arabidopsis thaliana, the predicted wheat protein had a C-terminal extension with three potential glycosylation sites. Northern analysis revealed that wheat 1,3-[beta]-glucanase mRNA was up-regulated in Al-intoxicated roots, with highest expression after 12 h. The antibody to A6 1,3-[beta]-glucanase from B. napus cross-reacted with a 56-kD protein that was induced after 24 h. A second partial cDNA clone showed similarity to genes encoding cytoskeletal fimbrin-like (actin-bundling) proteins. Although well studied in animals and fungi, fimbrins have not previously been described in plants. Fimbrin-like transcripts were up-regulated after 24 h of Al treatment in the Al-sensitive wheat cv Victory. In the Al-tolerant cv Atlas 66, fimbrin-like mRNA was up-regulated within 12 h by Al concentrations that did not inhibit root growth. Cellular stress associated with Al toxicity therefore causes up-regulation of a defense-related gene and a gene involved in the maintenance of cytoskeletal function.
... PR proteins that are induced by pathogen infection have been reported in wheat (Li et al. 2001), grapevine (Hatmi et al. 2013; Renault et al. 1996), mango (Ebrahim et al. 2011), banana (Harish et al. 2009), European plum (El-kereamy et al. 2011), and apple (Li et al. 2014). β-1,3-glucanases are PR-2 proteins that catalyze endotype hydrolytic cleavage of 1,3-β-D glycosidic linkages in β -1,3-glucans present in the cell walls of many pathogenic fungi (Simmons 1994). Induction of β-1,3-glucanase has been reported in various plants infected with fungal pathogens including tomato (Joosten and De Wit 1989), wheat (Kemp et al. 1999), peach (Zemanek et al. 2002), rubber tree (Thanseem et al. 2005), apple (Venisse et al. 2002), rucola (Gupta et al. 2013), V. flexuosa (Ahn et al. 2012), and strawberry (Fang et al. 2012). ...
... PR proteins that are induced by pathogen infection have been reported in wheat (Li et al. 2001), grapevine (Hatmi et al. 2013;Renault et al. 1996), mango (Ebrahim et al. 2011), banana (Harish et al. 2009), European plum (El-kereamy et al. 2011), and apple (Li et al. 2014). β-1,3-glucanases are PR-2 proteins that catalyze endotype hydrolytic cleavage of 1,3-β-D glycosidic linkages in β-1,3-glucans present in the cell walls of many pathogenic fungi (Simmons 1994). Induction of β-1,3-glucanase has been reported in various plants infected with fungal pathogens including tomato (Joosten and De Wit 1989), wheat (Kemp et al. 1999), peach (Zemanek et al. 2002), rubber tree (Thanseem et al. 2005), apple (Venisse et al. 2002), rucola (Gupta et al. 2013), V. flexuosa (Ahn et al. 2012), and strawberry (Fang et al. 2012). ...
... The degradation of fungal cell walls requires diverse enzymes, including b-1,3-glucanases and chitinases. A loss in the integrity of fungal cell walls results in a decrease in turgor pressure and a substantial reduction in virulence (Kang et al. 2019;Mauch et al. 1988;Simmons 1994). In this study, genes encoding b-1,3-glucanases (three DEGs) and chitinase (two DEGs) were differentially expressed in response to RsPV2-BJ infection (Table 1). ...
Rhizoctonia solani partitivirus 2 (RsPV2), in the genus Alphapartitivirus, confers hypovirulence on Rhizoctonia solani AG-1-IA, the causal agent of rice sheath blight. In this study, a new strain of RsPV2 obtained from R. solani AG-4HGI strain BJ-1H, the causal agent of black scurf on potato, was identified and designated as Rhizoctonia solani partitivirus 2 strain BJ-1H (RsPV2-BJ). An RNA sequencing analysis of strain BJ-1H and the virus RsPV2-BJ-free strain BJ-1H-VF derived from strain BJ-1H was conducted to investigate the potential molecular mechanism of hypovirulence induced by RsPV2-BJ. In total, 14319 unigenes were obtained, and 1341 unigenes were identified as differentially expressed genes (DEGs), with 570 DEGs being down-regulated and 771 being up-regulated. Notably, several up-regulated DEGs were annotated to cell-wall-degrading enzymes, including β-1,3-glucanases. Strain BJ-1H exhibited increased expression of β-1,3-glucanase following RsPV2-BJ infection, suggesting that cell wall autolysis activity in R. solani AG-4HGI strain BJ-1H might be promoted by RsPV2-BJ, inducing hypovirulence in its host fungus R. solani AG-4HGI. To the best of our knowledge, this is the first report on the potential mechanism of hypovirulence induced by a mycovirus in R. solani.
... The enzyme b-1,3-glucanases generally found in bacteria, fungi, and higher plants. The enzyme has the efficiency of degradation of the cell wall of fungi, yeasts and further classified as either exo or endo-b-1,3-glucanases (b-1,3-glucan: glucanohydrolase) (Pang et al., 2004;Simmons, 1994). Glucanase participates in cell wall degradation and further penetration into the host mycelium. ...
Injudicious application of chemical fertilizers in world has a considerable negative impact on economy and environmental sustainability. There is a growing need to turn back to nature or sustainable agents that promote evergreen agriculture. Potassium is important and well known constraint to crop production. Plant growth promoting microbes (PGPMs) promote the growth of the plants by diverse mechanisms such as they make the availability of various nutrients to the plants such as potassium, phosphorus, zinc, they produce various plant growth regulators, protect plants from pathogens, helps the plants to survive under stress conditions. A wide range of potassium solubilizing microbes have been reported viz, Acidithiobacillus, Agrobacterium, Arthrobacter, Aspergillus, Bacillus, Burkholderia, Enterobacter Pantoea, Flectobacillus, Klebsiella, Microbacterium, Myroides, Paenibacillus, Pseudomonas, and Stenotrophomonas. The present chapter deals with diversity of potassium solubilizing from diverse habiats, mechanism of potassium solubilization and mobilization, and their potential biotechnological applications for sustainable agriculture and environments. K-solubilizing PGP bacteria may have exploited as agricultural agent for wheat crop under different stresses condition, critical diseases and may have also used in the amelioration of K-deficient soils
... β-1,3-glucanase play a major role in plant defense response and belongs to PR-2 family of pathogenesis-related proteins. It is also present in fungi, bacteria, yeast, insects, fish and actinomycetes (Pan et al., 1989) and is involvedin the cleavage of β-1,3-glucan by breaking down β-1,3-glucosidic bonds (Simmons, 1994). β-1,3-glucan is an important structural component of cell wall in many pathogenic fungi but major constituent is chitin (Adams, 2004). ...
Fungi are historically notorious pests that have threatened availability of quality food. Several invasive species have appeared to be destructive for valuable crop species and even led to famine in certain severe cases. Surveillance and eradication of these disastrous microbial invaders is dependent on their sentinel behavior. Molecular Biology has helped to great extent in understanding these epidemic agents. Plant defense system as well as plant microbe interaction have well been explored and proved quite fruitful in understanding metabolic pathways involved in pathogenesis and defense response. Ultimately, researchers are able to define strategies for the control of these invasive pathogens. Genome editing has successfully been employed to develop pathogen resistant crops. Antifungal proteins have been expressed through transgenic technology to develop disease resistant plants. A few have proved to be the real success stories whereas others are at the stage of infancy. This review is an update about research work accomplished to-date, for the characterization and identification of fungal pathogens, metabolic pathways activated during plant pathogen interaction, advancements in the detection of fungal pathogens and transgenic plants developed to withstand pathogen attack.
... Moreover, RT-PCR expression analysis revealed that the PR2 expression increased in the resistant and susceptible genotypes over the inoculation time points, with the highest expression (6.4 and 1.99-fold for Banteng and WI2291, respectively) observed at 6 dpi. PR2 encodes for a 1,3-ß-glucanase (Simmons, 1994), belonging to the glycoside hydrolases family (Opassiri et al., 2010). 1, 3-ß-glucanase hydrolyses the ß-O-glycosidic bond of ß-glucan in plant cell walls, resulting in cell wall loosening and expansion (Akiyama et al., 2009). ...
Net blotch (NB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres, substantially reduces barley grain yield and quality worldwide. The role of salicylic acid (SA) signaling in NB resistance has been poorly documented. In this study, SA levels as well as the expression of the SA-responsive gene PR2 were monitored in infected leaves of two barley genotypes, Banteng (resistant) and WI2291 (susceptible), at different time points of infection. SA signaling was activated in bothgenotypes 24 hours post infection (hpi) as compared with non-inoculated plants. However, with or without pathogen pretreatment, SA signifi cantly increased (P=0.001) in Banteng comparing with WI2291. RT-PCR analysis revealed that PR2 expression increases in the resistant and susceptible genotypes over the inoculation time points, with maximum expression (6.4 and 1.99-fold, respectively) observed 6 dpi. PR2 expression was paralleled by an increase in leaf SA content as shown by the test coincidence (F 3 , 32 = 4.74, P = 0.001). Based on barley genotype resistance levels, our data strengthen the idea that SA signaling and PR2 play a role in barley NB reduction
... Moreover, RT-PCR expression analysis revealed that the PR2 expression increased in the resistant and susceptible genotypes over the inoculation time points, with the highest expression (6.4 and 1.99-fold for Banteng and WI2291, respectively) observed at 6 dpi. PR2 encodes for a 1,3-ß-glucanase (Simmons, 1994), belonging to the glycoside hydrolases family (Opassiri et al., 2010). 1, 3-ß-glucanase hydrolyses the ß-O-glycosidic bond of ß-glucan in plant cell walls, resulting in cell wall loosening and expansion (Akiyama et al., 2009). ...
Net blotch (NB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres, substantially reduces barley grain yield and quality worldwide. The role of salicylic acid (SA) signal-ing in NB resistance has been poorly documented. In this study, SA levels as well as the expression of the SA-responsive gene PR2 were monitored in infected leaves of two barley genotypes, Banteng (re-sistant) and WI2291 (susceptible), at different time points of infection. SA signaling was activated in bothgenotypes 24 hours post infection (hpi) as compared with non-inoculated plants. However, with or without pathogen pretreatment, SA significantly increased (P=0.001) in Banteng comparing with WI2291. RT-PCR analysis revealed that PR2 expression increases in the resistant and susceptible genotypes over the inoculation time points, with maximum expression (6.4 and 1.99-fold, respectively) observed 6 dpi. PR2 expression was paralleled by an increase in leaf SA content as shown by the test coincidence (F3, 32 = 4.74, P = 0.001). Based on barley genotype resistance levels, our data strengthen the idea that SA signaling and PR2 play a role in barley NB reduction.
... The higher activities of the selected defense genes such as PR2 and the higher level of SA in infected Banteng leaves tissue compared with the susceptible genotype WI 2291 may explain its high level of resistance. PR2 encodes 1,3-β-glucanase throughout the plant kingdom (Simmons, 1994) and belongs to the glycoside hydrolases family (Opassiri et al., 2010). 1,3-β-glucanase hydrolyses the β-O-glycosidic bond of β-glucan in plant cell walls, causing cell wall loosening and expansion (Akiyama et al., 2009). ...
Powdery mildew, caused by Blumeria graminis f. sp.
hordei (Bgh) is a common foliar disease of barley worldwide.
To better understand barley mechanisms to resist
this disease, compatible/incompatible barley-Bgh interactions
and some marker genes involved in salicylic acid
(SA) pathway were evaluated using qRT-PCR across four time
points after pathogen challenge. Data showed that
SA level contents of tolerant and susceptible genotypes
increased one day post inoculation (dpi) in comparison
with non-inoculated plants, and that the tolerant genotype
contained three-fold of total SA than the susceptible one.
This might be evidence that SA plays a positive role in
signaling events during fungal infection by Bgh. In general,
our study shows a remarkable discrepancy in the expression
pattern of those marker genes between compatible
and incompatible barley-Bgh interactions. The results of
qPCR revealed that the defense-related genes PR2, PAL
and LSD1 were expressed in the tolerant genotype over
the inoculation time points with 3.8, 4.11 and 3.16-fold
increases after 6 dpi. Moreover, the increase in gene expression
of the genes was closely related to the activation
of SA levels. Taken together, our results provide an insight
into the signaling pathway that accounts for classical gene
expression changes elicited during barley-Bgh interactions.
... β-1,3-Glucanases [EC 3.1.1.6] are widely spread in bacteria, fungi, and higher plants (Simmons 1994). This enzyme has interesting and important physiological roles and practical applications in the degradation of cell wall in fungi, yeasts, and higher plants (Pang et al. 2004). ...
Microbial community in the rhizosphere produces a variety of hydrolytic enzymes that are responsible for the degradation of various components of fungal pathogens. The extracellular hydrolytic enzymes excreted by soil rhizobia degrade cell wall components of plant pathogenic microbes. The enzymes of these types are able to breakdown glycosidic linkages present in the polysaccharide of the cell wall of phytopathogens. In this regard, plant growth-promoting rhizobacteria (PGPR) are known to colonize rhizosphere and enhance plant growth through different mechanisms that include (i) plant growth promotion and (ii) biological control of plant disease. Plant growth promotion mechanisms include mineralization of insoluble substances, production of plant growth hormones, biological nitrogen fixation, and promotion of root growth. Biocontrol mechanism involves competition, antibiosis, parasitism, induction of systemic acquired resistance (SAR), induction of systemic resistance (ISR), soil suppressiveness, and production of various antifungal metabolites; hydrolytic enzymes such as chitinase, glucanase, protease, and cellulase; and antibiotics such as 2,4-diacetyl phloroglucinol (DAPG), amphisin, oomycin A, hydrogen cyanide, phenazine, pyoluteorin, pyrrolnitrin, cyclic lipopeptides, oligomycin A, zwittermicin A, kanosamine, and xanthobaccin. Production of hydrolytic enzymes by PGPR is an important mechanism directed against phytopathogens for sustainable plant disease management. These enzymes break down the cell wall of fungal pathogens causing cell death. This review focuses on the different aspects of various hydrolytic enzymes produced by rhizoflora and their role in sustainable biocontrol of phytopathogens.
... b-Glucanases (PR2) have diverse activity and localization (Simmons, 1994). PpGns4 is a 1,3-b-D-glucanase expressed at higher levels in DR-than in DS-which may also contribute to the resistance of DR-. ...
The worldwide-distributed leaf peach curl disease is caused by the biotroph Taphrina deformans. To characterize the plant-fungus interaction, resistant and susceptible Prunus persica genotypes grown in the orchard were studied. Asymptomatic leaves were tested for fungal presence. In all resistant leaves analyzed the fungus was not detected. Conversely, leaves from the susceptible genotype were categorized according to the presence or absence of the pathogen.
Comparative metabolomic analysis disclosed the metabolite composition associated with resistant and susceptible interactions, and of compounds involved in fungal growth inhibition such as chlorogenic acid, whose in vitro antifungal activity was verified in this work.
Differential proteome studies revealed that chloroplasts are important site of plant defense responses against T. deformans. Members of the Bet-v1-like family protein differentially responded to the pathogen. Extracellular pathogenesis-related proteins, evaluated by qRT-PCR, and an enone oxidoreductase are constitutively present in leaves of resistant trees and could be related to fungal resistance.
This study is a global view of the changes in the metabolome, proteome and transcripts related to plant defense in naturally infected leaves of susceptible plants during the asymptomatic stage. Additionally, it provides clues to the successful molecular mechanisms operating in resistant plants, which neither develop the disease nor harbor the pathogen.
... Fungi have cross-linked chitin and glucan molecules as structural units of cell wall that are prone to degradation by hydrolytic enzymes such as chitinases and glucanases produced by plants. Chitin molecules consists of N-acetyl-D-glucosamine polymers having b-1,4-linkages that are hydrolyzed by chitinases, whereas the glucan molecule consists of b-1,3-glucosidic linkages that are cleaved by b-1,3-glucanase (Simmons 1994). This phenomenon has been widely utilized in genetic engineering to induce the overexpression of genes encoding hydrolytic enzymes. ...
Being sessile organisms, plants are continuously challenged by phytopathogenic fungi, contributing the largest share in loss due to plant disease. Plants naturally possess a well-developed and programmed protein-based defense system, capable of producing antimicrobial cationic peptides to ward off pathogen attack. Numerous genes encoding antifungal proteins have been isolated, cloned, sequenced and transgenically expressed against multiple phytopathogenic fungi successfully. Genetic engineering technology has been widely utilized to produce transgenic plants with enhanced resistance against pathogens. Pathogenesis-related proteins (PR-proteins) is a group of the most important inducible defense-related antifungal proteins, including defensins, thionins, osomtin-like proteins, thaumatin-like proteins, chitinases, glucanases, oxalate oxidase or oxalate oxidase-like proteins and lipid transfer proteins. Transgenic plants have been developed by imparting the artificial expression of genes encoding antifungal PR-proteins. The expression of transgenes belonging to a single group of PR-proteins or synergistic action of transgenes from different groups has greatly uplifted the level of defense response in plants against fungi. Transgenic expression of antifungal PR-proteins has led to remarkably enhanced resistance in transgenic plants. In this review, we have summarized the role of PR-proteins in plant defense against fungi and 15 years of success achieved so far to generate a variety of transgenic plants resistant against fungi through overexpression of transgenes from different groups of PR-proteins.
... β-1,3-Glucanases are widely spread in bacteria, fungi and higher plants [13]. The enzyme degrades cell wall of fungi, yeasts [14] . ...
Hydrolytic enzymes produced by rhizospearic microbes inhibit the growth of phytopathogens through hydrolysis of their cell wall, proteins and DNA. Hydrolytic enzyme producing microbes play more promising and sustainable role in controlling phytopathogens vis-à-vis chemicals fungicides. The huge number of microbes has been found as promising biocontrol agent.
... They include the transfer of phospholipids from liposomes or microsomes to mitochonderia (Breu et al., 1989), the transport of cuticular components required for the biosynthesis of surface waxes (Sterk et al., 1991;Meijer et al., 1993;Hollenbach et al., 1997), as well as the inhibition of fungal and bacterial pathogens of plants (Terras et al., 1992;. For example, plant chitinases (PR-3, PR-4, PR-8 and PR-11) or glucanases (PR-2) secreted to cell wall and extracellular space, generates chitosan oligomers or 3,4-ß-glucan oligomers from the pathogen cell wall, that elicit phytoalexin (Stintzi et al., 1993;Simmons 1994). PR-1 was found to be also localized in extracellular spaces (Niderman et al., 1995). ...
... β-glu proteins have molecular masses in a range from 33 to 44 kDa [10,11], and a molecular mass of 41 kDa in Hevea brasiliensis [12]. These proteins are hydrolytic enzymes that can cleave 1,3-β-D linkages in β-1,3-glucans, which are cell wall components in several pathogenic fungi [13,14] and are often the major components of a cell wall [15,16]. For example, Phytophthora, in the Class Oomycete, has 80 -90 % β-1,3-glucan in its cell wall. ...
The β-1,3-glucanase (β-glu) gene belongs to pathogenesis-related protein family 2 (PR2), which is induced by pathogens. Leaf fall disease caused by Phytophthora palmivora is the most serious disease affecting Para rubber (Hevea brasiliensis Müll. Arg.) seedlings. In this study, we examined the development of necrotic lesions and the molecular responses shown by H. brasiliensis clones RRIT 251 and RRIM 600 from infection by P. palmivora. The expansion of necrotic lesions on Para rubber leaves was observed around points of inoculation. The expression of β-glu was analyzed by RT-PCR, and the accumulation of β-1,3-glucanase protein was determined by denatured SDS-PAGE. The inoculation tests suggested that RRIT 251 clones have a better resistance than RRIM 600 clones, with infection occurring in 33.34 and 100% of inoculations, respectively. RNA analysis by RT-PCR demonstrated that both RRIT 251 and RRIM 600 clones expressed the β-glu gene during P. palmivora infection, and protein analysis by denatured SDS-PAGE displayed no obvious differences between the clones. Although expression of the β-glu gene occurred in both clones, the clones differed in phenotype. The better resistance of RRIT 251 relative to RRIM 600 against infection by inoculation may be associated with other defense mechanisms against Phytophthora infection.
... (1,3)--Glucan endo-hydrolases, enzymes that catalyse callose degradation, are also present in large gene families in plants (e.g. 50 genes in Arabidopsis), and are involved in various developmental processes such as germination, pollen tube growth regulation and microspore maturation (reviewed in Leubner-Metzger, 2003;Simmons, 1994), as well as in defence processes through their activity as pathogenesis-related (PR) proteins (reviewed in Leubner-Metzger and Meins, 1999). It is still unknown which members of this large gene family function under which circumstances and in which tissues; however, based on microarray data, 44 genes from this family were grouped into 13 expression clusters (Doxey et al., 2007). ...
This chapter discusses the cytology of the (1)lucan (callose) in plasmodesmata and sieve plate pores. Plasmodesmata (Pd) are co--xial membranous channels that cross walls of adjacent plant cells, linking the cytoplasm, plasma membranes and endoplasmic reticulum (ER) of cells and allowing direct cytoplasmic cell--o--ell communication of both small molecules and macromolecules (proteins and RNA). Transport through Pd mediates many processes in plants, among them information transfer for coordination of development, movement of photosynthesis products from mature to developing and storage tissues, responses to pathogen infection and systemic gene silencing. A basic structure of simple primary Pd consists of two coaxial membrane tubes. The inner membrane along the Pd axis, termed the desmotubule, is continuous with and connects the ER of the bordering cells; the outer coaxial membrane is continuous with and connects plasma membranes of adjacent cells. Between the membranes is a sleeve that interconnects the cytoplasm of the neighboring cells. Within the cytoplasmic sleeve are particles whose identity is still unknown, but have been interpreted to be cytoskeletal proteins.
... In addition to plant, β-1, 3-glucanases have been found in yeasts, actinomycetes, bacteria, fungi, insects, and fish [71,72].β-1,3-glucanases are able to catalyze the cleavage of the β-1,3-glucosidic bonds in β-1,3-glucan [73].β-1,3-glucan is a another major structural component of the cell walls of many pathogenic fungi [74,75]. For example, Phytophthora infestans is an oomycete pathogen that causes late blight of potato and tomato. ...
When resistant plants recognize cognate or matching elicitors, intracellular signal transduction pathways are activated that ultimately result in the derepression of a battery of genes called defense response genes. These latter genes encode producing various pathogenesis related (PR) toxic proteins such as chitinases, glucanases, lysozyme-active proteins, or cell wall strengthening proteins such as hydroxyproline rich glycoproteins. Response proteins may also be enzymes in biosynthetic pathways for lignification of cell walls or the production of phytoalexins, low molecular weight toxic chemicals that antagonize the invader. In the following portion, biochemical response of plant defence mechanism related to PR-protein including chitinase and glucanase, in addition to plant lignin content will be explained
... Previous studies have reported that cellulases are widely distributed in plants, 23 microbes, 24 and invertebrates that feed on marine algae. 2,6,7 In the present study, a cellulase that efficiently hydrolysed CMC-Na was purified to homogeneity by ammonium sulfate precipitation, SP-Sepharose cationic exchange and Sephacryl S-200 HR gel filtration column chromatographies from crude extracts of abalone hepatopancreas. ...
Background:
In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. Results A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two steps of column chromatographies. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting (PMF) analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-β-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerization of 2-4, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed a, a-diphenyl-b-picrythydrazyl (DPPH) free radical as well as hydrogen peroxide scavenging activity.
Conclusion:
It is feasible and meaningful to utilize cellulase from the viscera of abalone for preparation of functional oligosaccharides.
... Certaines protéines PR sont des hydrolases, telles que les chitinases et chitinases-lysozymes (Simmons, 1994;Rohini et Rao, 2000) et les glucanases (PR-2) (Grover et Gowthaman, 2003 ), capables de dégrader directement les parois de champignons et de bactéries. De plus, les chitinases et les inhibiteurs de protéase (PR6) peuvent aussi cibler des nématodes et les insectes herbivores (Van Loon et al., 2006). ...
... Plant (1,3)--glucanases form a highly diverse family of hydrolytic enzymes. Differences can be found in gene expression (Cabello et al., 1994;Garcia-Garcia et al., 1994;Romero et al., 1998;Xu et al., 1992), in their chemical structure, and in their subcellular localization (Simmons, 1994). Pathogen attack or environmental stress can induce (1,3)--glucanase transcript accumulation (Kaufmann et al., 1987;Simmons et al., 1992). ...
This chapter focuses on callose deposition by plants in response to pathogen attack. The deposition of the linear (1,3)-β-glucan callose is involved in several fundamental processes of plant development. However, particular attention has been focused on the formation of callose-containing papillae (cell wall thickenings of plants) in response to microbial attack. Papillae are not regarded as a defense response that can completely stop pathogens, rather they are thought to act as a physical barrier to slow pathogen invasion. However, callose-rich papillae are deposited at sites of penetration whether or not penetration is successful and the pathogen gains entry into the host cells, bringing into question the importance of papillae in plant defenses. Arabidopsis thaliana is one of the best studied models for plant defense responses. Mutants of this plant lacking callose-rich papillae revealed a potential function for callose in modulating signaling via the salicylic acid pathway. Mechanisms regulating callose synthesis are largely unknown, and most data about regulation of callose synthesis are based on correlations rather than direct evidence.
... The substrate specificities of various -1,3 glucanase is different between classes. Class I and II are 50-250 times better in degrading -1,3 glucan substrate than class III and IV [29]. ...
Proteins encoded by the host plant induced under pathological or related conditions are termed pathogenesis-related proteins. These proteins display high-degree of pathogen specificity and are coordinated at the level of transcription. Induction of PR's when measured on a time scale is a late event and its effect on the early infection is limited. Application of chemicals or microbially derived metabolites that mimic the effect of pathogen infection induces both PR's and acquired resistance. Numerous Pathogenesis Related proteins have been detected in rice, wheat, maize, sorghum, barley, tomato, pearl millet, bean, chickpea, soybean, pepper, sunflower, carrot, pepper, grape vine, alfalfa, celery, rubber and many other plants. The localization and distribution of the PR is related directly to the method and nature of the pathogen infection. The PR's have been classified into various families based on the shared sequence homology. PR's can also be grouped into different classes based on the migration in the native PAGE, reaction with specific antisera and mRNA probes. PR's have also been classified based on the biological
... Phenylamonialyase were quantified according to methods of Burrell and Rees using L-phenylalanine (Burrell and Rees, 1974). Glucanase were quantified according to method of Simmons (1994) by using leminarin as substrate and chitinase was quantified by using crab shell chitin (Mabuchi, 2000). ...
This study was done to investigate biochemical, histological and molecular basis of resistance after a compatible and incompatible interaction between Sorghum varieties and Colletotrichum sublineolum. In the susceptible variety, the pathogen invaded all types of tissues but in resistance variety, pathogen invasion was redistricted. Localized cell death was obvious in cortical region of resistant variety that stopped further invasion of pathogen. Epidermis of susceptible variety ruptured shortly after pathogen inoculation and fungal bodies emerged whereas in resistant variety a long delay was noted in occurrence of same event. A steep increase in total phenolics and enzymes related to phenylpropenoid pathway was observed after pathogen challenge in resistant variety as compared to the susceptible one. Histochemical studies revealed heavy deposition of lignin, callose, suberin, phenolics and peroxidases in the resistant variety. These enhanced depositions reduced the pathogen invasion in the host plant. Similarly, RT-PCR analysis revealed higher expression levels of PR protein-genes in the resistant variety. This study presents the nature of a resistant host defense mechanism against a virulent pathogen and made us to integrate defense mechanism for development of resistant varieties in future.
... Genes coding for proteins important in cellular defence, stress response and detoxification (Table 1) include a β-1,3glucanase precursor (1.9), which has been shown to be induced by several stress factors (Simmons, 1994). A putative endochitinase (1.6) and PR1 protein were also induced by CsCl application (Table 2; Supplementary material). ...
Excessive caesium can be toxic to plants. Here we investigated Cs uptake and caesium-induced gene expression in Arabidopsis thalian. Accumulation was measured in plants grown for 5 wk on agar supplemented with nontoxic and up to toxic levels of Cs. Caesium-induced gene expression was studied by suppression-subtractive hybridization (SSH) and RT–PCR. Caesium accumulated in leaf rosettes dependent upon the external concentration in the growth media, whereas the potassium concentration decreased in rosettes. At a concentration of 850 μM, Cs plants showed reduced development, and withered
with an increase in concentration to 1 mM Cs. SSH resulted in the isolation of 73 clones that were differentially expressed at a Cs concentration of 150 μM. Most of the genes identified belong to groups of genes encoding proteins in stress defence, detoxification, transport, homeostasis and general metabolism, and proteins controlling transcription and translation. The present study identified a number of marker genes for Cs in Arabidopsis grown under nontoxic Cs concentrations, indicating that Cs acts as an abiotic stress factor.
... The recognized PR proteins have been extensively reviewed (Broekaert et al. 2000) and currently comprise 17 families of induced proteins (van Loon et al. 2006). These families include one each of 1,3glucanases (Simmons 1994;Saikia et al. 2005), proteinase inhibitors (Mosolov et al. 2001), one specific peroxidase (Lagrimini et al.1987;Ghosh 2006), the PR-1 family with unknown biochemical properties (Niderman et al.1995), the thaumatin-like PR-5 family (van Loon 1982), the birch allergen Betv1-related PR-10 family (McGee et al. 2001), defensins (Terras et al.1992), lipid-transfer proteins (LTPs) (García-Olmedo et al.1995), thionins (Bohlmann and Apel 1991) and other proteins including 2S storage albumins (Terras et al.1993) and ribosome-inactivating proteins (RIPs) (Nielsen and Boston 2001). ...
In recent years, a considerable number of studies have harnessed the power of genomics to decipher the role of pathogenesis‐related (PR) proteins in plant defence against various biotic and abiotic stresses. Chitinases are PR antifungal proteins expressed constitutively at low levels in plants and induced during biotic pressures and are demonstrated to be involved in the plant defence responses. Remarkable induction of chitinase enzymes by various abiotic agents (salicylic acid, jasmonic acid, ethylene and ozone) and biotic components (pathogens, insect pest, fungal cell wall components and oligosaccharides) is well demonstrated in plants. Several reviews on plant chitinase expression during host–pathogen interaction are available for annual species, whilst reports of their expression in tree species are limited to a few woody perennials: Populus, Pinus, Picea, Eucalyptus, Castanea and Pseudotsuga. The aim of this paper is to review the induction of chitinase during various stresses and developmental processes in forest tree species.
... This xylem sap study, in addition to identifying candidate pathogenicity F. virguliforme proteins, detected pathogenicity related soybean protein families (Table 4A). Beta-1,3-glucanase is a pathogenic related family 2 (PR-2) type protein known to be secreted upon pathogen attack and shown to be present in the xylem sap of pathogen infected plants [26,47,48]. This protein has the ability to inhibit the fungal growth by degrading b-1,3-glucans of the fungal cell wall [49,50]. ...
Background:
Sudden death syndrome (SDS) caused by the ascomycete fungus, Fusarium virguliforme, exhibits root necrosis and leaf scorch or foliar SDS. The pathogen has never been identified from the above ground diseased foliar tissues. Foliar SDS is believed to be caused by host selective toxins, including FvTox1, secreted by the fungus. This study investigated if the xylem sap of F. virguliforme-infected soybean plants contains secreted F. virguliforme-proteins, some of which could cause foliar SDS development.
Results:
Xylem sap samples were collected from five biological replications of F. virguliforme-infected and uninfected soybean plants under controlled conditions. We identified five F. virguliforme proteins from the xylem sap of the F. virguliforme-infected soybean plants by conducting LC-ESI-MS/MS analysis. These five proteins were also present in the excreted proteome of the pathogen in culture filtrates. One of these proteins showed high sequence identity to cerato-platanin, a phytotoxin produced by Ceratocystis fimbriata f. sp. platani to cause canker stain disease in the plane tree. Of over 500 soybean proteins identified in this study, 112 were present in at least 80% of the sap samples collected from F. virguliforme-infected and -uninfected control plants. We have identified four soybean defense proteins from the xylem sap of F. virguliforme-infected soybean plants. The data have been deposited to the ProteomeXchange with identifier PXD000873.
Conclusion:
This study confirms that a few F. virguliforme proteins travel through the xylem, some of which could be involved in foliar SDS development. We have identified five candidate proteinaceous toxins, one of which showed high similarity to a previously characterized phytotoxin. We have also shown the presence of four soybean defense proteins in the xylem sap of F. virguliforme-infected soybean plants. This study laid the foundation for studying the molecular basis of foliar SDS development in soybean and possible defense mechanisms that may be involved in conferring immunity against F. virguliforme and other soybean pathogens.
... The class I members with approximate size of 33 kDa are basic and localized in the cell vacuole and are found in tobacco, tomato, potato, and other plant species [81]. The class II and class III proteins are acidic proteins with average molecular weights around 34 to 36 kDa secreted into the extracellular space [82]. Antifungal activity has been observed only in class Iglucanases. ...
Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.
... β-1,3-Glucanases β -1,3-Glucanases are widespread in bacteria, fungi, and higher plants [24]. These enzymes are classified as either exo-β -1,3-glucanases or endo-β -1,3-glucanases (β -1,3-glucan: glucanohydrolase. ...
Trichoderma spp. generally grows in its natural habitat on plant root surfaces and therefore it controls root diseases in particular. Trichoderma spp. were shown to be very efficient producers of extracellular enzymes, and some of these have been implicated in the biological control of plant diseases The mycoparasitic potential of Trichoderma spp. is well established. This trait has often been utilized as a means of in vitro screening for biocontrol candidates There are several mechanisms involved in Trichoderma antagonism namely antibiosis whereby the antagonic fungus shows production of antibiotics; competition for nutrients; and mycoparasitism. This work reviews data on the characteristics, properties, and role of lytic enzymes (including chitinases, β-1,3-glucanases, and proteases of certain Trichoderma species used for suppressing causative agents of plant diseases. Lytic enzymes of mycoparasitic fungi of the genus Trichoderma, capable of suppressing a number of fungal phytopathogens that originate in air or soil, are reviewed.
... SA is synthesized via the phenylpropanoid pathway and the conversion of phenylalanine to trans-cinnamic acid by PAL is the first step of this pathway [50]. There were five putative b-1,3-glucanases in the avocado dataset (Table S4) and these may play a role in callose deposition in plant cells [51] or function as PR2 proteins that break down the cell walls of pathogens [52]. Two of these showed much higher transcript abundance (.10 fold) when the infected libraries were compared to control libraries. ...
Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.
... The beta-1,3-glucanases are abundant, highly regulated enzymes and widely distributed in seed-plant species (Simmons, 1994). Although the major interest in beta-1,3-glucanases (PR-2) stems from their possible role in the fruit defence mechanisms against fungal infection (El Ghaouth et al., 2003), these enzymes are also implicated in diverse physiological and developmental processes in plant including cell division, fruit ripening, seed germination, and responses to wounding, cold, ozone and UV B. McCollum et al. (1997) the presence of both basic and acidic forms of beta 1,3-glucanase in grapefruit flavedo, where the basic isoforms of beta-1,3-glucanases represent the greatest amount of the total activity. ...
... and CHN (EC 3.2.1.14) are abundant proteins widely distributed in seed-plant species (for reviews, see Stone and Clarke, 1992;Simmons, 1994). Both enzymes have been implicated in responses to stress, wounding, and pathogen infection (Thalmair et al., 1996;for reviews, see Boller, 1988;Linthorst, 1991). ...
Class I isoforms of β-1,3-glucanases (βGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense. Tobacco (Nicotiana tabacum L.) βGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation. Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin. We report a novel pattern of βGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants. Abscisic acid (ABA) at a concentration of 10 μmmarkedly inhibited the induction of βGLU I but not of CHN I. RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of βGLU and CHN are induced in cell culture and that ABA inhibits steady-state βGLU I mRNA accumulation. Comparable inhibition of β-glucuronidase expression by ABA was observed for cells transformed with a tobacco βGLU I gene promoter/β-glucuronidase reporter gene fusion. Taken together, the results strongly suggest that ABA down-regulates transcription of βGLU I genes. This raises the possibility that some of the ABA effects on plant-defense responses might involve βGLU I.
Bread Wheat is one of the most important crops which has main position in nutrition of the world's population. Powdery mildew, caused by the biotrophic pathogen Blumeria graminis f.sp tritici (Bgt), known as a destructive disease of wheat worldwide. There are several methods to control the disease, but use of
inducers able to induce a systemic resistance in plants are very important. As of Piriformospora indica endomycorrhiza fungi and salicylic acid after symbioted with roots and inoculated on plants, respectively, with inducting systemic resistance can play a pivotal role in inducting resistance of plant to pathogens. Plants in response to biological agents produce numerous compounds such as reactive oxygen species,
phytoalexins and a group of proteins called pathogenesis related (PR) proteins. In order to examinate the
expression rate of PR1, PR2, PR3, PR5, PRX, PAL, NPR1, MLO and BI-1 genes using Real Time PCR
technique in response to Bgt and also change pattern of peroxidase, catalase and ascorbate peroxidase
enzymes, 40 Iranian commercial wheat genotypes were screened using Bgt in seedling stage. On the basis of
colonies number formed on samples, cv. Flat was selected as susceptible lines, and cv. Tajan and cv.
Gascojen were selecteted as as spring and winter resistance genotypes, respectively. For testing the ability of
fungus P. indica and SA for inducing resistance in wheat against Bgt fungus, cv. Falat treated seperately and
then exposed to Bgt fungi together with control plants. Similarly, to compare the pattern of genes
expression, cv. Falat together with resistant cultivars were exposed to fungus Bgt. The expression rate of
target genes was carried out at 5 time courses and in 3 independent replicates and then genes expression rate
were normalized in comparing with their relative internal reference Actin gene. Results indicated that in both
groups of treated and control plants, levels were increased after infection for all genes excepts MLO and BI-
1. Maximum expression level of genes were observed at 24 hours after infection. This process was observed
slowly in control plants but caused early and faster induction of plant defense genes in treated plants at early
hours after infection. At 48 hours after inoculatin, transcript levels of induced genes started to dampen in
both groups of experimental plants, indicating effective suppression of defense-associated genes upon
haustorium development. Also, comparing the expression patterns of genes in susceptible and resistant
cultivars showed that the expression of these genes in induced resistant varieties were much faster than the
Falat susceptible cultivar. In addition, the results of this study showed that the antioxidant activities of the
treated plants and resistant varieties are less than of the susceptible cultivar. Therefore, this phenomenon can
promoted the idea that these plants with reduced enzyme activities inducting the rapid cell death that is
combated with the pathogen. While symbiant plants except overexpression of peroxidase enzyme, were not
showed significantly different for other enzyme activitiesy than control plants. Therefore, the symbiotic
fungi seems can not be effective for inducing resistance via enzymatic pathway. Overall, results indicated
that Tajan and Gascojen cultivars with overexpression of resistance genes and antioxidant enzymes have
high potential for improving resistance to powdery mildew. Both of P. indica and SA inducers are able to
induce resistance in susceptible cultivar through overexpression of resistance genes and reducing number of
colonies grown on Bgt fungus p
Polyamines —(PAs) and proteins have been demonstrated to be fundamental for in vitro shoot development of Cedrela fissilis. We evaluated the influence of 6-benzyladenine (BA) and putrescine (Put) on the growth of shoots, PA metabolism and proteomic profiles of C. fissilis. The longest shoots were obtained under 2.5 µM BA + 2.5 mM Put treatment. The inhibition of Put synthesis by d-arginine (d-arg) reduced the activities of the Put biosynthesis enzymes arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and the endogenous contents of free Put, resulting in reduced shoot growth. The ODC activity was higher than that of ADC, being ODC the main enzyme in the synthesis of Put in C. fissilis. Inhibition of Put synthesis affected the proteomic profile, reducing the accumulation of the ubiquitin receptor RAD23c, peroxidase 15, ADP-ribosylation factor 1, ADP-ribosylation factor-like protein 8a, profilin-4, profilin-2, glucan endo-1,3-beta-glucosidase, and expansin-like B1 and increasing the accumulation of V-type proton ATPase catalytic subunit A and methionine gamma-lyase, highlighting the relevance of these proteins in promoting the length of shoots. Moreover, the transport protein SEC13 homolog B and the basic isoform glucan endo-1,3-beta-glucosidase, unique proteins in shoots treated with BA + Put, were related to the promotion of shoot growth. Our results show that the modulation of endogenous PAs and proteomic profiles is necessary to regulate in vitro morphogenesis in C. fissilis. Moreover, the ODC enzyme is highly involved in the synthesis of Put during in vitro shoot development and is described for the first time in this species.
The sections in this article are
Introduction
Phytoanticipins and Phytoalexins
Pathogenesis‐Related ( PR ) Proteins
Post‐Translational Modification, Intracellular Transport and Vacuolar Targeting of PR Proteins
Biotechnological Applications
Concluding Remarks
Plant growth and development is often challenged by several abiotic and biotic stresses, such as drought, cold, salinity, wounding, heavy metals, and pathogen attacks, respectively. A plant responds to these threats by activating a cascade of genes, encoding different effectors, receptors, and signaling and protective molecules. Among all, the induction and accumulation of pathogenesis-related (PR) proteins in plants in response to these adverse conditions is very important as PR proteins are an indispensible component of innate immune responses in plants under biotic or abiotic stress conditions. The PR proteins protect the plants from further infection by not only accumulating locally in the infected and surrounding tissues but also in remote uninfected tissues. Induction of PRs has been reported from many plant species belonging to different families suggesting a general role for these proteins in adaptation to biotic or abiotic stress conditions. PR proteins are also involved in hypersensitive response (HR) or systemic acquired resistance (SAR) against infection. Thus, PR proteins have been defined as “proteins encoded by the host plant but induced only in pathological or related situations,” the latter inferring situations of nonpathogenic origin. In this chapter, structure, biochemistry, source, regulation of gene expression, and role in defense mechanism of various pathogenesis-related proteins will be discussed.
Background:
Callose is a type of plant cell wall polysaccharide and is controlled by β-1, 3-glucanase and synthase. Abscisic acid (ABA) is an important plant hormone. Exogenous ABA induces the promotion of rice resistance to pests, and whether exogenous ABA could decline the loss of rice yield after brown planthopper (Nilaparvata lugens Stål; BPH) feeding is also the most important practical problems, whereas the mechanisms involved in rice resistance induced by ABA remain obscure.
Results:
Electronic penetration graph (EPG) recording further indicated a significant increase in rice resistance to BPH, and the number of eggs of BPH decreased significantly. With increasing concentrations of ABA, the loss of rice yield decreased significantly after BPH feeding. Further studies showed that β-1, 3-glucanase activity was significantly lower but the synthase activity was higher than the control after ABA treatments.
Conclusions:
Our results demonstrated that exogenous ABA suppressed β-1, 3-glucanase and induced synthase activity, which were beneficial for the promotion of callose deposition, which in turn is an important defence mechanism that prevented BPH from ingesting phloem sap. The studies could provide support for the insect-resistance mechanism after ABA treatment and provide a reference for the integrated management of other piercing sucking pests.
In agriculture, pathogens cause fungal diseases leading to loss in crop yield and quality. These diseases have been controlled by chemical fungicides. But their effects are often non-specific, targeting beneficial organisms as well as pathogens. The actual crop cultivars have disease responses and in-built genetic resistance against diseases for their defense against fungal pathogens. For instance, chitinases and β-glucanases have been proposed to have a role in fungal cell wall lysis by targeting key macromolecular components of the cell walls, i.e. chitin and β-glucan microfibrils. Chitin does not occur in plants, whereas in many plant-pathogenic fungi chitin comprises of 22–44 % cell wall material and maintains the structural integrity of hyphae. The glucans are structural polymers for maintaining rigidity and conferring protection.
Wheat is one of the food products that has an important place in the nutrition of human societies. The disease of powdery mildew in wheat caused by Blumeria graminis f.sp. tritici (Bgt) is one of the most important diseases of wheat. Plants exposed to biological agents produce a variety of compounds such as reactive oxygen species, phytoalexins, and a group of proteins called pathogenic proteins (PR). In present study, the expression pattern of PAL gene and a number of pathogen-related genes including PR1, PR2 and PR3 were investigated using qPCR technique in two genotypes resistant and susceptible to Bgt disease. Initially, 40 different commercial and native Iranian wheat genotypes were screened at the seedling stage after infection with wheat blight disease in terms of susceptibility based on the number of colonies grown per unit area to this disease. Finally, Tajan and Falat cultivars were selected as resistant and susceptible cultivars, respectively, and were exposed to Bgt fungus. Then, the expression of the desired gene was evaluated in five time courses in three replications and the amount of relative expression in comparison with the reference gene was normalized. The results showed that the expression of the studied genes in resistant and susceptible cultivars, after infection with powdery mildew, increased compared to the control time. Consideration of the trend showed that the amount of betaine of these genes after infection in Tajan cultivar increased faster than Falat cultivar. In both susceptible and resistant cultivars, the highest expression of genes was 24 hours after infection to prevent the infiltration and establishment of the fungal hostorium in the host cell. The maximum expression of all genes in resistant cultivar was significantly higher than susceptible cultivar, which according to these results, along with the main genes, intensify and maintain resistance. Both susceptible and resistant cultivars decreased gene expression 48 hours after infection, but the amount of reduction in susceptible cultivar Falat, was significantly higher than resistant cultivar Tajan. In addition, the changes of PR1 gene at the time of peak expression were more than other genes studied and Tajan cultivar with a 2.2-fold increase showed a significant difference compared to Falat cultivar that this gene seems to be in the pathogen-plant interaction, it plays an important role.
Abstract
Bread wheat is one of the most important food crops which has main position in nutrition of
the world's population. The production of this crop were always been faced to a variety of
challenges. Powdery mildew, caused by the biotrophic pathogen Blumeria graminis f.sp tritici
(Bgt), known as a destructive disease of wheat worldwide. There are several methods to control
the disease, but use of inducers as of SA, able to induce a systemic resistance in plants are very
important. In order to examination the ability of SA for inducing resistance in wheat against Bgt
fungus, Flat was selected as susceptible cultivar and treated with SA and then exposed to Bgt
fungi together with control plants. Then examine the expression rate of PR1, PR2, PR3 and PR5
genes using Real Time PCR technique in response to Bgt was carried out at 5 time courses and
in 3 independent replicates. Results indicated that in both groups of treated and control plants,
levels of gene expression were increased after infection for all genes. Maximum expression
level of genes were observed at 24 hours after infection. This process was observed slowly in
control plants but caused early and faster induction of plant defense genes in treated plants early
hours after infection rather than control plant. At 48 hours after inoculation, transcript levels of
induced genes started to dampen in both groups of experimental plants, indicating effective
suppression of defense associated genes upon haustorium development. Overall, results
indicated that SA inducer beacause of able to induce resistance in susceptible cultivar through
overexpression of resistance genes and reducing number of colonies grown on Bgt fungus per
unit area can one of the candidate of induced resistance in susceptible cultivar.
Keywords: Gene Expression, Induction Resistance, Salicylic Acid, Wheat
Mango (Mangifera indica L.) is known as “king of fruits” in India. More than 1000 mango varieties are currently cultivated in Indian Sub-continent. However most of the orchards of mango are infected with mango malformation disease (MMD), which every year leads to huge losses in yield of mango in range of 40 to 80 % in India. Till date there is no effective control measure against MMD. Floral Malformation, in contrast to vegetative one, is very virulent and can cause the loss of the entire crop. In the present study, six mango cultivars commonly grown in Gujarat, and all infected with various degrees of MMD were taken for studying their molecular relatedness, pathogen load and defense responsiveness via gene expression to rate whether hybrids or landrace among mango cultivars are better equipped to fight MMD. Genetic diversity analysis was performed using 30 SSR markers in order to bring out clustering pattern among the six cultivars belonging to orchards of Balisana and Prantij, Gujarat. The diversity analysis gave clues to the existence of wide genetic base among the six cultivars. Fungal load studies using Real Time PCR lead to the ranking of cultivars based on maximum and minimum infection load of pathogen. Absolute quantitation studies found that cultivars like Totapuri, Neelam and Amrapali were more resistant to MMD than highly popular cultivars like Kesar. The six mango cultivars were further quantified for pathogen responsiveness with 21 defense responsive genes using Real Time PCR. Among the 21 genes selected for the study, 11 genes were directly part of defense responsive pathways like Phenyl propanoid pathway and jasmonic acid pathway. Gene expression studies aided in ranking mango hybrid like Amrapali having better systemic acquired resistance response as 11 defense responsive genes were found upregulated in this cultivar followed by landrace Neelam which is in fact a parental line of Amrapali. If MMD remains unchecked it may lead to evolution of more virulent strains of Fusarium; propelling devastating consequences in mango cultivation. Hence mango hybrids developed via molecular and expressional screening will fasten process of establishment of resistant mango cultivars.
Previous evidence suggested the potyviral frame-shift protein P3N-PIPO is required for efficient viral intercellular movement. Host proteins are essential for virus to establish successful infection, many virus proteins play key roles in the viral infection cycle by interacting with that. Here, a yeast two-hybrid screening analysis was completed using Soybean mosaic virus (SMV) P3N-PIPO as the bait and a cDNA library from soybean infected with SMV as the prey to characterize the function of SMV P3N-PIPO. Fifty-four genes were isolated and analyzed by BLAST tools. Several genes encoding proteins that interacted with SMV P3N-PIPO were screened, including genes for inhibitory and transcription factors and those related to defense, transport, and photosynthesis. Some genes encoded proteins involved in metabolic activities in the chloroplast, such as photosystem I subunit PsaD and Calvin cycle protein CP12-2. Some genes were associated with defense responses, such as pathogenesis-related protein 1-like protein, stress-related protein-like protein, and stress enhanced protein 2. Other genes encoded defense-related transcription factors, such as WRKY transcription factor 51, while others were related to signal transduction, including a translationally-controlled tumor protein homolog, calcium-transporting ATPase 12, plasma membrane-type calcium-transporting ATPase 12-like protein, and calcineurin B-like protein 1-like isoform X1. Some genes coding for proteins related to affect viral plasmodesmata tracking, such as glucan endo-1,3-β-glucosidase (acidic isoform GL153). This study is the first to preliminarily delineate the interactions between SMV P3N-PIPO and host proteins related to defense responses during SMV infection.
Bread wheat is a crop that has the most important position in feeding human population. The powdery mildew disease in wheat caused by the agent Blumeria graminis f.sp. tritici (Bgt) is known as an deleterious wheat disease. Plants treated by biological agents will produce various compositions such as active oxygen species, phytoalexins, and a group of proteins known as the proteins relevant to virulent (PR). In present study the gene expression feature for PAL and a number of virulent genes including PR1, PR2, and PR3 were evaluated using qPCR in two sensitive and resistant genotypes to Bgt. First of all, 40 different Iranian commercial and local wheat genotypes were screened at seedling stage after inoculating by wheat powdery mildew disease for determining of the amount of sensitivity based on the number of grown colonies per unit area. Finally, two cultivars Tajan and Falat were selected as resistant and sensitive cultivars, respectively and then inoculated by Bgt fungus. Then after, the defined genes expression were considered using five time courses in three replications and their relative expression were normalized in comparison with the aktin control gene. Results showed that the amount of considered genes in sensitive and resistant cultivars after inoculating by the agent of powdery mildew have increasing trend in comparison with the control time. Consideration of the changes trend of expression demonstrated that the amount of expression of these genes after inoculating had faster increasing in resistant Tajan cultivar than that of sensitive Falat cultivar. In both sensitive and resistant cultivars, maximum expression of gene has been produced at 24 h after inoculation because prevention of the fungus hostorium penetration and fixation in the host cell. Maximum expression of all genes in resistant cultivars were more than that of sensitive cultivars
so that in attention to these results these genes together with the major genes can accelerate and protect from resistant. Both of resistant and sensitive cultivars at 48 h after inoculation showed reduction of expression, however, the amount of this reduction in Flat cultivar were significantly more than that of Tajan. Furthermore, changes in PR1 gene in the maximum expression time were more than that of the other studied genes and Tajan cultivar with 2.2 times increasing had significant differences with Falat cultivar, that showed this gene has serious important role in disease - plant interaction.
This research analysed the response of onion (Allium cepa) and A. fistulosum against Fusarium oxysporum f. sp. cepae (Foc) isolates and the associated changes in peroxidase, β-1,3-glucanase and chitinase activities. The response of A. cepa and A. fistulosum at different stages of seedling development were also evaluated. Several seedling tests were performed, and disease symptoms were evaluated 12-14 days after inoculation. Allium fistulosum behaved as more resistant than A. cepa cultivars by exposition to the most aggressive Foc isolates at sowing date. Increased levels of peroxidase and glucanase activities were found in the A. cepa and A. fistulosum seedlings exposed to the pathogen, and were positively correlated with disease symptoms. For chitinase activity, this correlation was found only for A. cepa. Two peroxidase isoforms were found to be specific for A. fistulosum roots after inoculation and could be involved in resistance. The inoculation at 7, 14 and 42 days after sowing showed that both host species were resistant to Foc, proving that onion susceptibility decreased promptly after germination. However, an increase in peroxidase and glucanase activities in 7-and 14-day-old inoculated seedling was detected only for A. cepa, suggesting an earlier acquisition of resistance in A. fistulosum.
Winter wheat landraces and modern Slovak cultivars were inoculated with the pathogen Fusarium culmorum Sacc. by spraying in May 2008, in plot experiments under natural conditions in Pieany, Slovakia. The objective was to examine the responses of the tested genotypes to inoculation with F. culmorum and to determine changes in the β-D-glucan content in the kernels. The area under the disease progress curve (AUDPC), Fusarium-damaged kernels (FDK) and the β-D-glucan and deoxynivalenol (DON) contents in the grains were determined using Megazyme and Ridascreen® Fast DON assay kits. Wheat landraces had lower AUDPC and FDK, and accumulated 67.4% less DON than modern cultivars. There were highly significant correlations (P < 0.01) between AUDPC and DON content, between FDK and DON, and between AUDPC and FDK. The correlation between β-D-glucan content and AUDPC was also significant (P < 0.05), but not correlations between β-D-glucan and other traits. The β-D-glucan content in the grain of wheat genotypes artificially inoculated with F. culmorum was lower than in grains without infection. The wheat landraces contained more β-D-glucan than modern cultivars and showed higher resistance to F. culmorum. The three wheat landraces had significantly lower spike and kernel infection compared to modern cultivars and could be used to breed elite cultivars with enhanced Fusarium head blight resistance.
The Aspergillus flavus infection of peanut (Arachis hypogaea) results in the accumulation of aflatoxins in seeds, which are very harmful to humans and animals. Mutation breeding programs are an effective way of inducing resistant mutants. In this study, we induced a genetic variation by using ethyl methanesulfonate (EMS) and gamma rays treatment for four peanut cultivars (32 mutants from Giza 6, 22 mutants from Gregory, 15 mutants from Giza 4 and 15 mutants from Giza 5). The resistant mutants for A. flavus were identified by analyzing β-1-3-glucanases activities of the controls and infected mutants using polyacrylamide gel electrophoresis (PAGE). Two, four and four mutants derived from Giza 6, Gregory and Giza 4, respectively, showed high activities of β-1-3-glucanases and therefore more resistant to the infection of A. flavus. The genetic similarity of these mutants and their controls was also tested using random amplified polymorphic DNA (RAPD) approach. Although natural polymorphism among peanut cultivars was very low, RAPD patterns showed high polymorphism percentage of DNA fragments (37.13%).
Cereal coleoptile cell walls have exo- and endoglucanases capable of mediating the hydrolysis of non-cellulosic β-(l,3)(l,4)-glucan in situ. A purified exoglucanase (EC 3.2.1.58) resolved as a single band at 73.5 kDa, while endoglucanase isozymes consistently appeared as two bands at 32.9 and 34.3 kDa when subjected to SDS-PAGE. HPLC analysis of the native proteins by gel-permeation chromatography revealed molecular weights of ca. 55 and 29 kDa for the exo- and endoglucanases, respectively. The exoglucanase has an isoelectric focusing point at pi 7.2 and the endoglucanase isozymes appeared as two major bands, one at pi 7.8 and another at 7.3. Deglycosylation of the native proteins followed by SDS-PAGE demonstrated that sugars accounted for ca. 6.5 % of the exoglucanase and were 12.5 and 8.8 % of the two endoglucanase isozymes, respectively. After deglycosylation the two endoglucanases converged at 30.0 kDa, suggesting polypeptide homology and that divergence in electrophoretic mobility was a consequence of glycosylation. Antibodies raised against intact exo- and endoglucanases recognized the polypeptide of the corresponding enzymes, irrespective of glycosylation. The N-terminal amino acid sequence supported the conclusion that the exo- and endoglucanase have different polypeptide structures.
The air pollutant ozone is a potent abiotic inducer of defense-related enzymes such as pathogenesis-related proteins. Here we report on the accumulation of ß-1,3-glucanase and chitinase in Nicotiana tabacum L. treated with ozone and ultraviolet B radiation, singly and in combination, under a simulated sunlight spectrum. Ozone (0.16 μL·L−1, 2 × 5h) induced the basic isoforms of ß-1,3-glucanase in both, ozone-sensitive (Bel W3) and -tolerant (Bel B) cultivars, while chitinase was only affected in cv. Bel W3. Ultraviolet B radiation (7.5 MED) alone did not lead to ß-1,3-glucanase or chitinase induction. In combined treatments ultraviolet B increased the ozone-dependent lesion formation and reduced chitinase accumulation in the sensitive cv. Bel W3. Analysis of the intercellular washing fluid of ozone-treated plants revealed the accumulation of a major ozone-related protein (O3R-1) of 28 kDa within 32 h. Microsequence analysis of two tryptic peptides showed 100 % homology to acidic chitinase PR-3b. These results indicate that basic ß-1,3-glucanase and chitinase are distinctly regulated in ozone and ultraviolet B treated tobacco, and that ultraviolet B radiation with a similar UV edge as the solar spectrum does not lead to an accumulation of basic pathogenesis-related proteins.
Here we describe the induction patterns of β-1,3-glucanase (βGlu; EC 3.2·1.39) in leaves of susceptible and resistant melon cultivars in response to infection by the cucurbit powdery mildew fungus Sphaerotheca fusca. Upon inoculation, βGlu activity increased more rapidly in the resistant cv. "PMR-6" than in the susceptible cv. "Rochet" by either spectrophotometric or gel assays. A single βGlu polypeptide of 33 kDa was detected by Western blot analysis in both cultivars, that was first detected in the resistant cultivar. Two-dimensional analysis of the 33 kDa βGlu polypeptides revealed that in both cultivars such bands were composed of two acidic polypeptides; one being common to both cultivars and the other specific to each cultivar. Furthermore, Northern blot analysis using an homologous βGlu cDNA probe isolated by RT-PCR from powdery mildew-infected leaves of the resistant cultivar, showed an earlier induction of βGlu transcripts in this cultivar than in the susceptible one. The role of this hydrolytic enzyme in melon plant defence against powdery mildew is discussed.
We report the identification of a full-length cDNA encoding an acidic class III β-1,3-glucanase (CrGlcQ) from the flavedo of the chilling-sensitive ‘Fortune’ mandarin. By using heterologous expression of the CrGlcQ cDNA in Escherichia coli, we showed that it encoded a protein with glucanase but not antifreeze activity. Accumulation of the CrGlcQ mRNA appears to be linked to chilling-induced damage occurring during holding of fruit at a chilling temperature (2°C). The CrGlcQ transcript was also up-regulated by mechanical wounding and by exogenous ethylene at low temperature, which reduced chilling injury (CI). This result suggests that the CrglcQ gene may play a role in reducing chilling-induced peel damage in citrus fruit. Furthermore, the induction of this gene in wounded fruit appears to be dependent on ethylene but also on an ethylene-independent signal associated with mechanical damage.
Many molecular biologists are turning their attention to the interactions between plant pathogens and their hosts. These offer some promising model systems for investigating the control of gene expression in plants and, of course, offer promise for the future genetic engineering of crop plants to incorporate desirable traits such as disease resistance. Similarly, many plant pathologists are adopting the techniques of molecular biology to solve problems that have proved intractable by conventional biochemical and physiological methods of investigation. The problems encountered by both groups are somewhat different and we hope that this article will be of interest to all in the field by providing a review of current work, while attempting to explain many of the specialist terms involved.
When cloned pith and leaf tissues of Nicotiana tabacum L. cv. Havana 425 are subcultured for 3 d on auxin-containing medium and labelled for 18 h with [(35)S]methionine, up to 10% of the labelled, soluble-protein fraction is found in a single band with an apparent molecular weight of approx. 32,000-34,000 dalton on sodium-dodecylsulfate polyacrylamide-gel electrophoretograms. The labelling of this band, designated P33, is dramatically inhibited by the cytokinin, kinetin, in some cell lines at concentrations as low as 1.4·10(-8) M. P33 is a major component of the protein fraction obtained from non-habituated clones, cytokinin-habituated clones, and revertant subclones of crown-gall-transformed clones, but cannot be detected in clones habituated for both auxin and cytokinin, or crown-gall-transformed clones. The evidence supports the hypothesis that cytokinin in the presence of auxin regulates the production of a specific, major polypeptide in the soluble-protein fraction of the tissue and that this protein is not produced in tissues autotrophic for both auxin and cytokinin.
Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129-134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml(-1) was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders.
We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3-β-glucosidase (β-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 μl·l(-1), 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic β-1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and β-1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.
A single pulse of O3 (0.15 microliter per liter, 5 hours) induced beta-1,3-glucanase and chitinase activities in O3-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O3-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for beta-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O3-tolerant cultivar Bel B,beta-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O3 concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in beta-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O3 treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of beta-1,3-glucanase and chitinase activities following O3 treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O3 treatment increased the amount of the beta-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O3 levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.
Two genes encode (1-->3,1-->4)-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes EI and EII in barley (Hordeum vulgare L.). Specific DNA probes have been used in Northern analyses to examine the developmental regulation of individual (1-->3,1-->4)-beta-glucanase genes in the aleurone and scutellum of germinated grain and in young leaves and young roots. In aleurone and scutella excised from germinated grain, mRNAs encoding both isoenzymes are present but developmental patterns differ between the two tissues. Thus, levels of both isoenzyme EI and EII mRNA increase significantly in the aleurone between 1 and 3 days after the initiation of germination. In the scutellum, isoenzyme EI mRNA predominates and decreases as germination proceeds. Isoenzyme EI mRNA appears in young leaves approximately 8 days after the initiation of germination and levels rise until about 20 days. Enzyme activity in leaf extracts parallels the development of isoenzyme EI mRNA. No isoenzyme EII mRNA is detected in the leaves in this period. Analysis of RNA from different leaf segments indicates that the isoenzyme EI mRNA is distributed relatively evenly along the length of the leaf. In young roots, mRNA encoding (1-->3,1-4)-beta-glucanase isoenzyme EI is detected at high levels 3 to 6 days after the initiation of germination; again, little or no isoenzyme EII mRNA is found. Overall, transcription of the (1-->3,1-->4)-beta-glucanase isoenzyme EII gene appears to be restricted to the germinating grain, whereas isoenzyme EI is expressed in a wider range of tissues during seedling development.
The acidic, extracellular, glucan endo-1,3-t8-glucosidases (EC 3.2.1.39; j0-1,3-glucanases), pathogenesis-related proteins-2,-N, and-O (i.e. PR-2, PR-N, and PR-0) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-0. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of ,B-1,3-glucan-ase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the fl-1,3-glucan-ase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of ,-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.
Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.
Excised Zea mays L. embryos were cultured on Linsmaier and Skoog medium. Coleoptiles were sampled at regular intervals and the length, fresh weight, cell wall weight, and cell wall neutral sugar composition were determined. A specific β-d-glucanase from Bacillus subtilis was used to determine the content of a (1 → 3),(1 → 4)-β-d-glucan.
Coleoptiles elongated through the 5th day following imbibition with the most rapid elongation occurring between days 3 and 4. The greatest net rate of incorporation of cell wall per coleoptile occurred between the 2nd and 3rd days when deposition of approximately one-third of the maximum net glucan level was observed. By day 5, the amount of glucan present had increased 34-fold from the 6 micrograms per coleoptile on day 1 and accounted for about 14% of the cell wall (w/w). Thereafter, the glucan content declined until only 3.3% (w/w) remained by day 10. In this 10-day interval, xylose increased 32% and cellulose content doubled, while proportions of other neutral sugars changed less dramatically.
These results are consistent with a possible role for the β-d-glucan in elongation of the Zea coleoptile. Moreover, changes in the quantity of this wall component clearly reflect the dynamic nature of plant cell wall polysaccharides. An evaluation of glucan dynamics in vivo suggests that in vitro autolysis studies employing Zea coleoptile walls may overestimate the actual rate of glucan turnover in the intact tissue.
Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glu-canase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-l,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [3H]chitin, as well as in their relative lysozyme activity. Similarly, the two 0-1,3-glu-canases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin. © 1988 American Society of Plant Biologists. All rights reserved.
When isolated aleurone layers of barley are incubated they produce a number of hydrolytic enzymes which can be divided into three groups. The synthesis and the secretion of the hydrolases in the first group is greatly enhanced by gibberellic acid. Using the density labeling technique of Filner and Varner (3) it has been shown that the increase in enzymatic activity of two of the enzymes in this group (a-amylase and protease) is due to de novo synthesis (3, 6). The marked effect of gibberellic acid on the rate of enzyme synthesis makes this an ideal system to study hormonal control of protein synthesis (10). The total enzymatic activity of hydrolases in the second group (e.g., ribonuclease and B-glucanase) does not show the same response to GA. There is a considerable increase in enzymatic activity during imbibition of the seeds, but the addition of GA to the isolated aleurone layers causes only a small increase in the total amount of enzyme activity (2, 7). However, release of these enzymes in the medium is dependent on GA, and this system has been used to study hormonal control of enzyme secretion (2, 7). Whether or not the increase in enzymatic activity which occurs during imbibition of the halfseeds and incubation of the aleurone layers is due to de novo synthesis has never been determined. Finally, the enzymatic activity of at least one hydrolase, /3-amylase, increases in the presence of GA, but this is due to release of preformed enzyme and not to de novo synthesis (5). We now present evidence consistent with the idea that two enzymes in the second group are also synthesized de novo.
Avena coleoptile sections were treated with a fraction of a fungal filtrate containing a potent cellulase. Elongation rate was not affected although turgor pressure remained constant and wall extensibility was increased. These data show that the simple weakening of cell walls is not sufficient to promote growth and suggest that endogenous polysaccharidases are not the means by which the growth rate of the coleoptile is regulated.
A high level of activity of a β-1,3-glucan hydrolase is present in leaves of Nicotiana glutinosa and the enzyme is also present in the roots, midribs, petioles and stems. By comparison, very low levels of β-1,4-glucan hydrolase are found throughout the plant. The activity of the β-1,3-glucan hydrolase in leaves aged on the plant was found to increase 14-fold during the course of leaf senescence and to reach a maximum in yellow-green leaves. Detached leaves and leaf discs floated on water in the dark showed similar patterns of change.
The increase in β-1,3-glucan hydrolase activity during senescence is apparently not due to the loss of an inhibitor from young green leaves or to the formation of an enzyme activator in yellow leaves. The enzyme in yellow leaves was electrophoretically indistinguishable from that in green leaves. The hydrolase is not firmly attached to the cell walls and is not present in the particulate fraction sedimenting at 105400xg for 60 min. Within the leaf cell it is therefore likely to be located either in the cytoplasm or in an easily disrupted structure such as a vacuole.
The relationship of the hydrolase to leaf senescence was investigated by examining the effect of plant hormones on the changes in level of hydrolase, protein and chlorophyll in leaf discs during senescence. IAA (10 μM) and GA3 (50 μM) did not alter the normal patterns of change, whilst Kin (50 μM) delayed the loss of protein and chlorophyll and also delayed and decreased the rise in hydrolase activity. In contrast, ABA (190 μM) which increased the rate of loss of protein and chlorophyll, also caused a decrease in the rate and extent of the rise in hydrolase.
Possible functions of the hydrolase in the leaf are discussed.
A diallel cross analysis of gum content in barley (Hordeum vulgare) was made using six cultivars of two-rowed spring barley as parents. A Jinks-Hayman analysis of F2 progeny means showed that gum content was controlled by a simple additive-dominance genetic system and that low gum content was strongly dominant. The analysis suggested that gum content was principally controlled by two or three genes showing a high degree of dominance. Some genotype-environment interaction was detected in a comparison between the F2 and F3 generations which were grown in different years and locations. However, the character was found to be highly heritable both within and between generations, suggesting that the selection and breeding of barleys of reduced gum content should not be difficult.
The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 μg/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.
Molecular-sieve chromatography of the cell-wall protein resolved endo-β-D-glucanase and exo-β-1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo-β-D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.
Cell-wall β-D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze β-D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of β-D-glucan in vitro.
During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall β-D-glucan, the primary substrate for autolytic reactions.
A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.
Accumulation of messenger RNAs in potato tuber discs was analysed during the hypersensitive response induced by treatment with the biotic elicitor arachidonic acid. In vitro translation of polysomal poly(A)(+) RNAs indicated that the accumulation of some sixteen mRNAs varied following treatment with arachidonic acid, and that the level of thirteen of these was increased. Two cDNA closes (pSTH-1 and-2) were isolated from a library of elicitor-treated tissue cDNAs. Northern blot analysis using these clones as molecular probes indicated that the levels of at least two mRNAs were markedly increased after elicitor treatment. In hybrid-released translation experiments, each of the cDNA clones selected more than one mRNA. Translation of these mRNAs yielded two polypeptides of Mr 45 000 (for the pSTH-1 clone), and three polypeptides of Me 17 000 (for the pSTH-2 clone). The low molecular weight polypeptides may correspond to potato pathogenesis-related (PR) proteins.
A comparison has been made of the relative effectiveness of light quality and quantity and gibberellic acid (GA3) treatment on the elongation growth of the coleoptile and the first foliage leaf in durum wheat (Triticum durum Desf. cvs. Cappelli and Creso). The cultivar Creso is a shortstrawed variety carrying the Gai 1 gene on chromosome 4A, which influences both plant height and insensitivity to applied gibberellins. The main conclusions are as follows: 1) coleoptile elongation growth appears to be modulated via the fluencerate-dependent action of a blue-light receptor and via a low energy response of phytochrome; 2) the inhibition of first-foliage-leaf growth depends on the operation of a single blue-light-responsive photoreceptor; 3) high energy blue light produces the same inhibitory effect on the two wheat cultivars, whereas at relatively low fluences of white and blue light, the cultivar Creso is more sensitive; 4) the insensitivity to applied GA3 exerted by the gene Gai 1 in Creso is independent of light; 5) in Cappelli, the action of light on coleoptiles appears to be independent of the applied GA3, whereas the hormone is able to change the pattern of growth inhibition of the first-foliage-leaf.
A library of complementary DNA (cDNA) clones has been prepared from polyadenylated RNA (poly(A)(+)RNA) from auxin (2,4-dichlorophenoxyacetic acid)-treated soybean (Glycine max (L.) Merr. cv. Wayne) seedlings. Using differential hybridization, four clones were selected as auxin-responsive, and characterized. The levels of the RNA sequences homologous to the cDNA clones were examined in the hypocotyl of the intact seedling and in excised hypocotyl sections before and after auxin treatment, using RNA blot hybridization analysis. RNA levels are rapidly increased (within 0.25-0.5 h) following auxin treatment and the response in the hypocotyl of the intact seedling is transient, reaching maximum RNA levels 2-4 h after auxin application. Increases in RNA levels were also observed with the auxins indole 3-acetic acid and 2,4,5-trichlorophenoxyacetic acid, but not with the ethylene-producing compound, Ethephon (2-chloroethylphosphonic acid). Hybridization analysis of in-vitro transcription products made in nuclei isolated from untreated and auxin-treated soybean primary leaves and excised hypocotyl sections indicates that, for the two cDNA clones analyzed, the increased RNA levels in auxin-treated organs are at least partially the result of increased transcriptional activity of specific DNA sequences.
A highly sensitive and specific "rocket" immunoassay was used to measure the content of an endo-type β-1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of β-1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, α-naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l(-1) NAA and 0.3 mg·l(-1) kinetin are transferred onto medium without hormones or with either hormone added separately, the β-1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is β-1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This β-1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.
Plastic and elastic in-vitro extensibilities (E pland E el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E pl (ΔE pl ) is strictly correlated with the growth rate for a period of 3-4 h. Subsequently, ΔE plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that ΔE plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the ΔE plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of ΔE pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. ΔE el is not affected by either IAA, ABA, or CHI. It is concluded that δE pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514-520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.
Expression sites of genes encoding (1→3,1→4)-β-glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A(32)P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (1→3,1→4)-β-glucanase is detected in ungerminated grain. Expression of (1→3,1→4)-β-glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (1→3,1→4)-β-glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (1→3,1→4)-β-glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.
Ethylene treatment (approx. 20 μl ·1(-1) in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo-β-1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10(-5) M α-naphthaleneacetic acid) and cytokinin (1.4 · 10(-6) M kinetin). Evidence is presented that cultured tobacco cells require ethylene for β-1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl2; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1(-1) norbornadiene in air) inhibited β-1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of β-1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces β 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.
Mutation breeding has been used to improve the speed of germination in the high-yielding spring barley variety Troubadour. Five mutants were selected which combined fast germination and good agronomic performance. Two of these mutants yielded significantly more than did Troubadour over eight environments, and showed a clear improvement in their malting quality through an increase in extract yield. The improvement in malting quality appeared to be due to a decrease in the β-glucan content, which seemed to enhance the germination speed and thus the starch degradation. The improvement in grain yield is postulated to be due to a better early growth caused by the enhanced germination speed. All the described changes could theoretically be explained by a single mutation event in each of the mutant genotypes, affecting the quantity of β-glucans present in the endosperm.
Ethylene induced chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and β-1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [(35)S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the β-1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native β-1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and β-1,3-glucanase. A putative β-1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of β-1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for β-1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and β-1,3-glucanase are regulated co-ordinately at the level of mRNA.
A variety of mutants altered in hormone biosynthesis and response pathways have been described. Genetic and molecular analyses of these mutants have contributed information on the control of plant developmental processes and on mechanisms of signal transduction. A sampling of hormone mutants and their role in elucidating modes of hormone action in higher plants is described.
An endoglucanase liberated from Zea mays seedling cell-walls by LiCl was purified by using SP-Sephadex, CM-Sephadex, and gel filtration, resulting in a 98-fold increase in specific activity. It has a pH optimum of 4.5–5.0 and is heat stable up to 40–45°. Compounds that interact with sulfhydryl groups did not inhibit the activity of the enzyme, nor did EDTA, suggesting that the enzyme does not require free sulfhydryl groups or metal ions for activity. The endoglucanase has an apparent molecular weight of 20–25,000 and an isoelectric point of ⩾9. Hydrolytic activity against (1»3),(1»4)-β-d-glucans is restricted to isolated sites, with the release of high-molecular-weight products (10–15,000). Action on isolated, inactivated Zea cell-walls caused the release of approximately the same products as observed when the enzyme was incubated with soluble (1→3), (1→4)-β-d-glucans.
Nutrient availability in relation to broiler chick performance was evaluated in diets containing barley, oat groats or wheat with β-glucanase supplementation. Both growth and feed conversion of chicks (0–3 weeks) were improved significantly with enzyme supplementation for diets containing hulled or hulless barley or oat groats. No response to dietary enzyme was evident for chicks given wheat diets. Added dietary fat (1.0, 4.5 or 8.0%) also improved the performance of chicks fed on wheat or hulled barley, but results were variable when fat was added to hulless barley or oat-groat diets without enzyme. Both amino-acid and starch utilization in chicks fed on hulless barley or oat-groat diets were improved with enzyme supplementation, however, the response was less than that seen for fat digestibility. Most of the dietary fat was not derived from the cereal source indicating that β-glucan affected nutrient availability of the entire diet.
The influence on the productive value of diets for broiler chickens based on barley harvested at two stages of ripeness, when including β-glucanase in increasing amounts, was evaluated in a production experiment over three weeks. Feed consumption and live weight increased (P < 0.001) and feed conversion was improved (P < 0.001) by including β-glucanase in the diets. Dry matter content of excreta increased significantly as a result of β-glucanase inclusion. Broiler chickens receiving diets based on barley harvested at combine ripeness were slightly heavier (P < 0.05) at three weeks than those fed on barley harvested at early yellow ripeness.
Sixteen barley cultivars selected for variation in extract viscosity were grown at five locations across western Canada. Differences in extract viscosity among locations were most apparent for high viscosity genotypes while those exhibiting low viscosity were more uniform across locations. Eight barley genotypes grown in a single location representing high and low viscosity traits were given to broiler chickens in a 3-week growth study. Chicks fed on low viscosity barley gained significantly faster (P < 0.01), and converted feed more efficiently (P < 0.01) than those given high viscosity barley. Two high and two low viscosity genotypes were given to chicks with or without β-glucanase (Aspergillus niger) supplementation. With no enzyme addition chicks given low viscosity barley surpassed those given high viscosity barley with respect to growth (P < 0.01) and feed conversion (P < 0.01). Enzyme supplementation improved chick performance with both barley types, however the response was greater for high viscosity barley. Performance of chicks fed on enzyme-supplemented diets was similar for high and low viscosity barleys.
Incubation of segments of Phaseolus aureus hypocotyls in an indoleacetic acid (IAA) solution (5 · 10−6 M) induces an increment of the fresh weight of the segments and simultaneously a decrease of the pH of the incubation medium. This can be observed with segments from all regions of the hypocotyl although there is a gradient in growing potential along this organ. The autolysis rates as well as the glucanase activities of cell walls originated from the “upper” and “lower” parts of the hypocotyls are compared. A possible intervention of cell wall β1 → 3 endoglucanasesin the auxin-inducedgrowth is discussed.
Glycoproteins were extracted from isolated cell walls of Phytophthora megasperma f.sp. glycinea (formerly P. megasperma var. sojae) with 0·1 n NaOH at 0·C and elicited glyceollin in soybean hypocotyls with the same specificity as the fungus races from which they were obtained. Fractionation of the crude extracts on DEAE Bio-Gel and Bio-Gel A-5m columns showed that specific elicitor activity was associated with the presence of high molecular weight glycoproteins detected by SDS gel electrophoresis. The glycoproteins appeared to contain only glucose and mannose as neutral sugars. The elicitor activity of the glycoproteins was not diminished by boiling at 100°C or pronase treatment, but was destroyed by periodate, thus indicating that the carbohydrate portions are important for activity. The glycoproteins were the only concanavalin A reactive species detected in the crude cell wall extracts, and fluorescein labelled concanavalin A was hapten-specifically bound to living hyphae of the fungus and to native but not NaOH-extracted isolated cell walls. Therefore it was concluded that the glycoproteins are present at the surface of the fungus cell wall. Tunicamycin, which inhibits the glycosylation of eucaryote surface glycoproteins, was a potent inhibitor of mycelial growth of the fungus. The data supported the hypothesis that race specificity in the soybean—P. megasperma f.sp. glycinea system may be determined by specific plant recognition of fungus surface glycoproteins.
β-(1,3)-Glucanase activity and the growth of Fusarium oxysporum f. sp. melonis in near-isogenic lines of resistant and susceptible muskmelon were studied. The activity of the enzyme increased in response to infection. However, in both hypocotyls and roots, the increase was greater (approx. 2 times) in the resistant (Hemed) seedlings than in the susceptible (On) ones. The activity was initiated by the infection process or could be induced by pretreatment with laminarin. Fungal colonization, as determined by chitin content, was correlated with the disease reaction in the cultivars tested. It was concluded that the increase in activity of the host β-(1,3)-glucanase in the resistant cultivar provides a potential defence mechanism against F. oxysporum f. sp. melonis induced wilt.
The pH-dependence of the kinetic parameters for the hydrolysis of yeast glucan with endo-(1→3)-β-d-glucanase II from Flav. dormitator var. glucanolyticae FA-5 suggests that two residues (histidyl and carboxyl) are involved in the enzyme action. Chemical modification of the enzyme has been studied in order to identify the kinds and the number of amino acid residues involved in enzyme action. Photo-oxidation and carbethoxylation of the enzyme indicated that the decomposition of a histidine residue is responsible for the loss of activity. Modification of the enzyme with Woodward's reagent K indicated that ∼ 12 carboxyl residues in the enzyme are involved in the catalytic and/or substrate binding-site.
SUMMARY Spraying tobacco plants with salicylic acid induces both the synthesis of 'pathogenesis-related' (PR) proteins and resistance to viruses that can induce necrotic lesions. We show that spraying Samsun NN tobacco with salicylic acid induced the production of PR-1 mRNAs and inhibited the systemic multiplication of alfalfa mosaic virus (A1MV) by 90%. Salicylic acid treatment also induced the synthesis of PR proteins in bean and cowpea plants, and reduced by 75 % the production of local lesions in A1MV-infected bean plants. Salicylic acid inhibited the replication of A1MV in cowpea protoplasts by up to 99%, depending on the mode of application. In A1MV- inoculated cowpea protoplasts, the production of viral minus-strand RNA, plus-strand RNA and coat protein was abolished, indicating that salicylic acid inhibits an early step in the A1MV replication cycle. The viability of the cells and the synthesis of host proteins were not affected by salicylic acid. Another aromatic compound, p-coumaric acid, induced neither PR proteins nor resistance to virus infection.
The release of soluble carbohydrates from isolated cell wall of maize (Zea mays L) was investigated in the range of pH 1 to 8.5. The pH profile demonstrated two peaks, a broad peak at pH 6 due to enzymatic breakdown of β-glucan to monosaccharides (wall autolysis) and a sharp peak at pH 2.5 due to acid-mediated, nonenzymatic liberation of macromolecular β-glucan from the wall. The pH dependence of acid-induced growth and cell-wall extensibility of coleoptile segments closely agrees with the pH dependence of acid-mediated β-glucan solubilization in the isolated wall. However, there is no evidence that enzymatic or nonenzymatic β-glucan solubilization is involved in the mechanism of auxin-mediated growth.
Soybean (Glycine max) β-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced β-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean β-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable β-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a λ-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with β-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of β-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean β-1,3-endoglucanase exhibited 53% amino acid homology to a β-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a β-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable β-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.
Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a′, a1, b, b′, and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a′, and a1 were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-β-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-β-glucanase and a′ and a1 reacted with anti-chitinase antibodies. Similarly, b and b′ comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti-osmotin antibodies. In addition it has been shown that 1,3-β-glucanase and chitinase activities increased at the same time as a, a′, and a1 accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins.
Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDN A-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, l→4)-β-glucanase genes is located on chromosome 1. © 1988 American Society of Plant Biologists. All rights reserved.
Increases in b-proteins were detected in controls only 6 days after challenge. A basal level of chitinases was always detected, but increases in chitinases above this level in immunized plants followed a profile similar to that of the β-1,3-glucanases and other b-proteins. It is likely that β-1,3-glucanases, chitinases, and other b-proteins are coordinately regulated in tobacco. The increases in these proteins coincided with the onset of immunization in plants injected with P. tabacina, and the levels were maintained during the period after challenge, when the development of P. tabacina was restricted
The tobacco hybrid Nicotiana glutinosa × Nicotiana debneyi is much more resistant than either parental species to tobacco mosaic virus, tobacco necrosis virus, Pseudomonas syringae pv. syringae, P. syringae pv. tabaci and Peronospora tabacina infections. While N. glutinosa is very susceptible to Cercospora nicotianae, Chalara elegans and Phytophthora parasitica var. nicotianae, the hybrid is as resistant as N. debneyi to these fungi. The resistance of the hybrid is linked to high levels of chitinase, β-1,3-glucanase, peroxidase (PO) and polyphenoloxidase (PPO). The parental species contain much lower levels of these enzymes unless they are locally infected with necrotizing viruses. In those cases, chitinase, β-1,3-glucanase and PO, but not PPO, increase in the whole plant to levels comparable with those found in the hybrid, and resistance to secondary infection develops (systemic acquired resistance). All the acidic isoenzymes of PO present in both parental species after infection are constitutively expressed in the hybrid. The activity of phenylalanine ammonia-lyase was similar in all plants. These results reinforce the hypothesis that hydrolases and PO are involved in resistance against various pathogens.
By using a two-dimensional polyacrylamide gel electrophoresis (PAGE) system, one major additional band with chitinase activity could be detected in Allium porrum (leek) root extracts after colonization by Glomus versiforme or Glomus intraradix when compared to control root extracts. After separation under native conditions in the first dimension, this band was observed both in the Davis system (designed to separate native acidic or neutral proteins) and in the Reisfeld system (designed to separate basic proteins). After the second dimension in sodium dodecyl sulfate (SDS)-PAGE under non-reducing conditions, its apparent molecular mass was determined to be about 30 kDa. In Glomus fasciculatum-colonized root extracts of Allium cepa (onion), four additional bands with chitinase activity were found. The same additional bands were obtained in onion roots colonized by the other Glomus species. Among the four bands, one of them was separated in the first dimension in both the Davis system and the Reisfeld system, two were only detected as acidic or neutral isoforms, while the last one was present as a basic isoform. Their apparent molecular masses were estimated at 33, 35 and 50 kDa. In vesicular-arbuscular mycorrhizae (VAM)-colonized pea root extracts, one acidic chitinase isoform was strongly stimulated while another acidic isoform was less highly induced in the various mycorrhizal interactions. Their apparent molecular masses were 30 and 47 kDA. Chitosanase activities were detected in leek and onion roots colonized by the different VAM fungi, while no chitosanase was observed in either non-mycorrhizal or VAM-colonized pea roots extracts. In VAM-colonized leek roots, the main chitosanase activity had an estimated apparent molecular mass of 20 kDa as determined by SDA-PAGE under non-reducing conditions. Endomycorrhizal onion roots exhibited three chitosanases with estimated apparent molecular masses of 14, 20 and 35 kDa, the two latter being induced by VAM fungal colonization. Finally, no induction of β-1,3-glucanase activity was detected in VAM-colonized roots of leek and onion. In pea roots, one β-1,3-glucanase activity was revealed, but it was not present in all VAM interactions.
NDP-glucose pyrophosphorylase activities were measured in different parts of growing mung bean seedlings. Amongst the different enzymic activities only UDP-glucose pyrophosphorylase proved to be active, showing highest activity in the lower, no-longer elongating, sections of the hypocotyl. In contrast UDP-glucose : glucan synthetase activity was highest in the upper, still-elongating sections of the hypocotyl. Using hypocotyls of 2–6-day-old seedlings it could be demonstrated that UDP-glucose pyrophosphorylase activity was increasing as elongation ceased. At the same time UDP-glucose : glucan synthetase activity was decreasing.
The activities of enzymes hydrolysing sodium carboxymethylpachyman, sodium carboxymethylcellulose and salicin have been measured in extracts from a number of higher plants. The properties of the β-1,3-glucan hydrolase system from xylem and phloem of grape vine canes have been investigated and its relationship to the seasonal removal of sieve tube callose explored.
Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall ß-D-glucan and their possible role in extension growth. An exo-ß-1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both ß1–4 and ß1–3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-ß-D-glucanase. Similarly, the capacity of the purified exo-ß-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-ß-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a ß-glucosidase and did not appear to participate in the hydrolysis of wall ß-D-glucan.
Plants can be systemically immunized against diseases caused by fungi, bacteria, and viruses by restricted infection with fungi, bacteria, or viruses. Immunization followed by a booster inoculation protects cucumber, watermelon, and muskmelon throughout the season, and a single immunization protects cucumber against at least 10 unrelated diseases.
Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.
