Plant cell walls as targets for biotechnology

Chapple, Clint; Carpita, Nick

doi:10.1016/s1369-5266(98)80022-8

Cited by 57 publications

(34 citation statements)

References 43 publications

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“…Chapple and Carpita (1998) suggested that membraneanchored EGases may proofread the glucan chains and excise disordered amorphous cellulose. The second possibility regards determination of chain length.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Molhoj

2001

Plant Physiology

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The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.

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Section: Discussionmentioning

confidence: 99%

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Molhoj

2001

Plant Physiology

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“…Prolonging the desirable texture during ripening is the key to increasing fruit shelf life (27). For texture analysis and shelf life determination, transgenic and control (only vector transformed/ nontransformed) fruits were harvested at the pink stage and stored at room temperature (23-25°C and 55-60% relative humidity).…”

Section: Never Ripe (Nrmentioning

confidence: 99%

Enhancement of fruit shelf life by suppressingN-glycan processing enzymes

Meli

Ghosh²,

Prabha³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

194

167

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In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have been made to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall-degrading proteins. However, these have met with very limited success. N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specific N-glycoprotein modifying enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed ≈2.5-and ≈2-fold firmer fruits in the α-Man and β-Hex RNAi lines, respectively, and ≈30 days of enhanced shelf life. Overexpression of α-Man or β-Hex resulted in excessive fruit softening. Expression of α-Man and β-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening, rin (ripening inhibitor). Furthermore, transcriptomic comparative studies demonstrate the downregulation of cell wall degradation-and ripening-related genes in RNAi fruits. It is evident from these results that N-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation of N-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield.

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“…putative cinnamoyl-CoA reductases (CCRs: JB116 and JB196) and CADs (F193, F138, and F122), enzymes performing the last committed steps in the biosynthesis of lignin (Chapple and Carpita, 1998).…”

Section: The Importance Of the Vascular System And Lignification In Tmentioning

confidence: 99%

Novel Insight into Vascular, Stress, and Auxin-Dependent and -Independent Gene Expression Programs in Strawberry, a Non-Climacteric Fruit

Keizer²,

et al. 2002

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Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria ϫ ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.

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Plant cell walls as targets for biotechnology

Cited by 57 publications

References 43 publications

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Enhancement of fruit shelf life by suppressingN-glycan processing enzymes

Novel Insight into Vascular, Stress, and Auxin-Dependent and -Independent Gene Expression Programs in Strawberry, a Non-Climacteric Fruit

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