Source: OKLAHOMA STATE UNIVERSITY submitted to
THE STRUCTURE OF PECTINS FROM COTTON CELL WALLS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0139153
Grant No.
(N/A)
Project No.
OKL02099
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Mort, A. J.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
Knowing the details of the structure of pectins will help explain their roles and behavior. Details of the structure of pectins and methods to detect them will be invaluable to scientists working on the biosynthesis of pectins. It will allow us to predict which are the most likely links that should be targeted by enzymes in the plant during growth, developmental processes such as fruit ripening, and response to pathogens. It will allow us to predict all of the enzymes that a saprophyte would need to digest the pectin into monomers. In addition, we will also be able to explain the commercially important rheological behavior of pectins, and how that is changed by enzymic and chemical modifications of the pectins. During our research, we will be continuing to develop methods that are applicable to oligo- and polysaccharide characterization in general. These should be useful to a wide range of scientists. We will ensure that there will continue to be at least a few researchers familiar with both plants and modern approaches to carbohydrate polymer analysis.
Animal Health Component
5%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20624991000100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
It is our expectation that the primary plant cell wall is a covalently crosslinked network of polysaccharides (and sometimes protein) analogous to the bacterial peptidoglycan network and the glucan-mannoprotein-chitin network in yeast. One goal of the project is to characterize the linkages between the various regions of pectin and between pectin and the other polymers. The other is to discover putative transglycosylases that form the linkages between the polymers. To find the crosslinkages we will use cloned (monospecific) enzymes to degrade the polymers and separate out fragments which retain characteristics of two different polymers. These will then be characterized by NMR and mass spectroscopy. We have isolated and characterized oligosaccharides representing the crosslinkages between homogalacturonan and rhamnogalacturonan (RG) and between galactans and arabinans and RG during the previous project period. It remains to be determined how xylogalacturonan and rhamnogalacturonan are linked together. We have also found that xyloglucan and RG are crosslinked via a highly branched 1-5 linked arabinan. We plan to characterize the connections between the arabinan and the xyloglucan and between the arabinan and the RG using the same fragmentation, separation, and characterization approach mentioned above. To find the transglycosylases we are planning to develop an assay using the recently developed BlotGlyco beads. These beads are made by polymerizing an acrylamide monomer to which hydrazine is attached. The high density of hydrazide groups allows reducing sugars to covalently attach to the beads. Oligosaccharides which are putative acceptors for the transglycosylases will be attached to the beads and then exposed to protein extracts from plants and potential glycosyl donors. After an incubation period all extraneous materials can be washed away and the beads interrogated with enzymes which should release fragments of attached donor. Since the hydrazide-sugar linkage can be reversed by warm mildly acidic conditions the acceptor oligomer with a fragment of the donor should be recoverable from the beads.
Project Methods
Commercial apple pectin, watermelon fruit pectin, and both cotton and arabidopsis culture cell wall pectin will be first degraded with EPG to remove the majority of the HG. They will then be carefully saponified with KOH and digested with either EPG with added potassium oxalate to digest almost all of the HG. Ultrafiltration, to remove the HG fragments, will result in RG-XGA complexes with galactan and arabinan sidechains along with residual stubs of HG. The molecular weight distributions of the complexes will be determined by size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS). Galactan sideschains will be mostly removed by digestion with arabinosidase, galactosidase, and endogalactanase. Arabinan chains will be mostly removed by arabinosidase and endoarabinanase. After ultrafiltration to remove small fragments, the molecular weight distributions will again be determined by SEC-MALLS. The RG will then be degraded with RGase and the products separated by ultrafiltration. Short oligomers of RG, a small percentage of which will have HG stubs attached, will be separated by PA1 chromatography. Each fraction will be surveyed by MALDI TOF MS for the presence of excess GalA over Rha. Those with extra GalA will be characterized by electrospray MS/MS and 2D NMR spectroscopy. What remains polymeric after the RGase digestion will be characterized by SEC-MALLS and then digested with XGAase. The products from the digestion will be separated on the PA1 column and the fractions surveyed by MALDI TOF MS. Those containing GalA, Rha, and Xyl will be characterized by electrospray MS/MS and 2D NMR spectroscopy. We are proposing to use oligosaccharides linked to BlotGlyco H beads as the acceptor for transglycosylation reactions. After incubation with suspected donors and tissue extracts the beads will be thoroughly washed free of non-linked polymers and then interrogated by enzymes which should cleave characteristic oligomers from the transglycosylated polymer. The released oligomers and the oligomers remaining bound to the beads will be characterized by CZE as APTS or ANTS derivatives or by MALDI TOF MS

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A post doc, Xiagmei Wu and a graduate student, Xiaoyu Qiao, are directly associated with the project. How have the results been disseminated to communities of interest? I communicated a report called Half of the xyloglucan in cell walls of tissue cultures is linked to pectin via a highly branched arabinan Fu J., Umezu S., Patel P., Mort A. The paper was selected for an oral presentation. The XIIIth Cell Wall Meeting, Nantes, France July 7-12 What do you plan to do during the next reporting period to accomplish the goals? I hope that we can learn to use LC MS/MS spectrometry to help determine structures of oligosaccharides. This should help us to complete our work on finding the mode of linkage between pectic regions and between pectin and xyloglucan. In collaboration with Rolf Prade, I hope that my students will finally successfully express large quantities of enzymes for testing their effectiveness in breaking down biomass.

Impacts
What was accomplished under these goals? We have continued investigating the structure of pectin, revisiting the use of liquid hydrogen fluoride to cleave specific glycosidic linkages. We have also almost completed determining the structure of Karaya gum by generating characteristic fragments by HF solvolysis for NMR and mass spectrometry. Karaya gum is an exudate from trees and is used as a food additive and in cosmetics. We looked at it because it is similar to pectin. We found that some of the previously proposed structure was wrong. We have cloned all of the sequences for enzymes we want to test in biomass conversion but not yet got them expressed in the fungal host for “large scale” production. Guru has cloned the promoter regions of three isozymes and fused them to GFP. We can now investigate by fluorescence imaging under what conditions the different enzymes are expressed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Mort A, Bell-Eunice G, Wu X: Characterization of a methyl-esterified tetragalacturonide fragment isolated from a commercial pectin with a medium degree of methyl-esterification. Carbohydr Res 2013, 380:108-111


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The structures of a mix of oligosaccharides representing junction zones between rhamnogalacturonan and xylogalacturonan, prepared as described in last years report were determined. We confirmed that a considerable fraction of the xyloglucan in arabidopsis walls is covalently linked to rhamnogalacturonan. We analyzed the results we obtained from microarray experiments designed to quantitate the changes in gene expression brought about by switching fungi from growth on glucose to growth on powdered sorghum stover as carbon source. We have started to clone several enzymes which appear to be key in overcoming the recalcitrance of cell walls to digestion. These include the "so called" glycosyl hydrolase 61 enzymes which appear to oxidatively cleave crystalline cellulose and glucuronoyl esterase which may cleave linkages between lignin and xylan. Results from the work on the linkages between regions of pectins was presented at the 3rd Biennial DOE Physical Biosciences Research Meeting, Bolger Conference Center, Potomac, MD, October 14-17, 2012 . Wu,X.,Mort, A.J. (2011) Structure of pectins. PARTICIPANTS: Andrew Mort, PI., Songyue Cheng, graduate student, Xiangmei Wu, post doctoral fellow, Soreiyu Umezu,part time post doctoral fellow, Jooel Otiko, graduate student, Cynthia Dobbs, graduate student, and Guru Jagadeeswaran, Senior Research Specialist. We have a strong collaboration on the cloning of enzymes with Rolf Prade of the Department of Microbiology and Molecular Genetics at OSU. TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. PROJECT MODIFICATIONS: The understanding of the process of cell wall degradation by fungi is receiving greater attention.

Impacts
The linkage between rhamnogalacturonan and xylogalacturonan appears to be linear with a simple transition from RG to XGA via an alpha 1-4 linked galacturonic acid. Enzymic digestion of the link between xyloglucan and rhamnogalacturonan from arabidopsis cell walls confirmed that it is via a highly branched arabinan just as it is in cotton. Out of 9505 total genes in Aspergillus nidulans 3664 genes showed at least a two fold change in expression level between growth on glucose vs growth on sorghum.

Publications

  • Ray A, Saykhedkar S, Ayoubi-Canaan P, Hartson SD, Prade R, Mort AJ: Phanerochaete chrysosporium produces a diverse array of extracellular enzymes when grown on sorghum. Appl Microbiol Biotechnol 2012, 93:2075-2089. Saykhedkar S, Ray A, Ayoubi-Canaan P, Hartson SD, Prade R, Mort AJ: A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover. Biotechnol Biofuels 2012, 5:52. Segato F, Damasio ARL, Goncalves TA, Murakami MT, Squina FM, Polizeli MdLTM, Mort AJ, Prade RA: Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber. Biotechnol Biofuels 2012, 5:21.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Oligosaccharides representing junction zones between rhamnogalacturonan and homogalacturonan, and between rhamnogalacturonan and xylogalacturonan were generated using cloned enzymes and isolated as was described in last years report. The structures of these oligosaccharides was investigated by NMR and MS/MS using a LTQ-Orbitrap XL hybrid mass spectrometer. We did more enzymatic investigation of the xyloglucan rhamnogalacturonan crosslink. A graduate student cloned six pectin-degrading enzymes from Aspergillus niveus, a thermophilic fungus. We expect that these will give us more flexibility in how we dissect the pectins so that we can determine more detailed structural information. Results from the work on the linkages between regions of pectins was presented at the Plant Biology 2011 meeting. Wu,X.,Mort, A.J. (2011) Abs # P02031: Primary structure model of pectin from plant cell wall. PARTICIPANTS: Andrew Mort, PI. Purvi Patel, graduate student, Songyue Chen,graduate student, Xiangmei Wu, post doctoral fellow, and Soreiyu Umezu,part time post doctoral fellow. We have a strong collaboration on the cloning of enzymes with Rolf Prade of the Department of Microbiology and Molecular Genetics at OSU. TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Studies of oligosaccharides joining rhamnogalacturonan and homogalacturonan indicate that the linkage is linear, not branched as has been proposed by Vincken, J.P., et al., Pectin-the hairy thing, in Advances in Pectin and Pectinase Research, A.G.J. Voragen, H.A. Schols, and R.G.F. Visser, Editors. 2003, Kluwer Academic Publishers: Dordrecht. p. 47-59. The linkage between rhamnogalacturonan and xylogalacturonan also appears to be linear. Enzymic digestion of the link between xyloglucan and rhamnogalacturonan confirmed that it is via a highly branched arabinan. The newly cloned pectinases do show subtle differences in their modes of action, so will be useful in our structural studies.

Publications

  • Mort, A., & Wu, X. (2011). Capillary electrophoresis with detection by laser-induced fluorescence. Methods Mol. Biol. (N. Y., NY, U. S.), 715, 93-102.
  • Wang, H., Squina, F., Segato, F., Mort, A., Lee, D., Pappan, K., & Prade, R. (2011). High-temperature enzymatic breakdown of cellulose. Appl. Environ. Microbiol., 77, 5199-5206.
  • Wu,X.,Mort, A.J. (2011) Abs # P02031: Primary structure model of pectin from plant cell wall. Plant Biology 2011.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Apple pectin was digested with a series of cloned mono-specific enzymes and the resulting oligosaccharides containing fragments of rhamnogalacturonan fractionated on a high performance anion exchange column. The molecular weights of each fraction were determined by MALDITOF mass spectrometry. Oligomers with novel structures were further purified by gel permeation chromatography and their structures determined using 2D-NMR spectroscopy. Xyloglucan-pectin complex obtained from cotton culture cell walls was purified by ion exchange chromatography, characterized and then digested with cloned arabinosidase and a thermo-tolerant endoarabinanase. The products were then subjected to fractionation by ion exchange chromatography. PARTICIPANTS: Andrew Mort, PI. Purvi Patel, graduate student, and Xiangmei Wu, post doctoral fellow. MS Purvi Patel graduated with a masters degree in Biochemistry and Molecular Biology. TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The oligosaccharides purified and characterized from the apple pectin had structures which reveal several linkages between different regions of pectin. One showed that, at least in some cases homogalacturonan is liked to the non-reducing end of rhamnogalacturonan. Another showed that long galactan chains link directly to rhamnose residues in the rhamnogalacturonan. Others showed that arabinan chains are linked to the rhamnogalacturonan via single galactose residues attached to rhamnose in the rhamnogalacturonan. Manuscripts describing the characterization of the oligomers are in preparation. Since the xyloglucan and rhamnogalacturonan could be separated from eachother after the combined arabinosidase and endoarabinanase digestion we are convinced that the link between xyloglucan and rhamnogalacturonan is via a branched arabinan.

Publications

  • Squina, Fabio M.; Santos, Camila R.; Ribeiro, Daniela A.; Cota, Junio; de Oliveira, Renata R.; Ruller, Roberto; Mort, Andrew; Murakami, Mario T.; Prade, Rolf A. Substrate cleavage pattern, biophysical characterization and low-resolution structure of a novel hyperthermostable arabinanase from Thermotoga petrophila. Biochemical and Biophysical Research Communications (2010),399(4),505-511.
  • Liu Z, Bhattacharyya S, Ning B, Midoro-Horiuti T, Czerwinski EW, Goldblum RM, Mort A, Kearney CM. (2010) Plant-Expressed Recombinant Mountain Cedar Allergen Jun a 1 Is Allergenic and Has Limited Pectate Lyase Activity. Int Arch Allergy Immunol;153:347-358.
  • Mort, A. J. and Wu, X. (2011) Capillary electrophoresis with detection by laser-induced fluorescence. In Methods in Molecular Biology: The Plant Cell Wall Methods and Protocols, Zoe A. Popper ed., Humana Press. In press.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Apple pectin was digested with cloned enzymes in the sequence: endoploygalacturonase (EPG), saponification, EPG, arabinosidase, galactanase, xylogalacturonase, and then rhammnogalacturonase (RGase). After each treatment oligomers were removed by ultrafiltration. The oligomers produced by the final RGase treatment were fractionated on ion exchange chromatography and by size on a Superdex peptide column. Oligosaccharides representing the junction between arabinan and rhamnogalacturan (RG), and galactan and RG were recovered. Their structures were determined by mass spectrometry and NMR spectroscopy. A complex between xyloglucan (XG) and RG was extracted with strong alkali from cotton cell walls after EPG digestion. XG that was not covalently linked to the RG was removed by repeated ion exchange chromatography. Digestion of the complex with cloned arabinosidase followed by endoarabinanase allowed separation of most of the XG and RG in the complex by ion exchange chromatography. PARTICIPANTS: Andrew Mort, PI. Purvi Patel, graduate student, and Xiangmei Wu, post doctoral fellow. MS Purvi Patel will graduate with a masters degree in Biochemistry and Molecular Biology in December. TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
From the structures of the oligosaccharides we purified and characterized from apple pectin we can deduce that beta- (1-4)galactans are linked to rhamnogalcturonans via a galactose linked to the 4- position of rhamnose in the RG backbone. However, alpha (1-5) arabinans are also linked to the RG backbone via a single galactose on the 4- position of rhamnose. This finding is contrary to most models of pectin widely reported in text books and reviews on the structure of pectin. These results will be helpful to researchers studying the biosynthesis of pectins. It was hypothesized almost thirty-five years ago that xyloglucan is crosslinked to pectin via an arabinan or arabinogalactan. since then there have been only a small number of reports that XG and pectin might be covalently linked together, and none on the nature of the linkage. We have shown almost conclusively that the linkage is via a branched arabinan.

Publications

  • Ishimaru M, Smith DL, Mort AJ, Gross KC (2009) Enzymatic activity and substrate specificity of recombinant tomato beta -galactosidases 4 and 5. Planta 229: 447-456
  • Squina FM, Mort AJ, Decker SR, Prade RA (2009) Xylan decomposition by Aspergillus clavatus endo-xylanase. Protein Expression & Purification 68: 65-71


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: PROGRESS: 2007/10 TO 2008/09 OUTPUTS: An optimal sequence of cloned enzyme digestions was developed and applied to pectin from apple fruit. The digestion products were separated by HPLC and analyzed for their sugar compositions by GLC, and their structures by mass spectrometry and NMR spectroscopy. The putative linkage between xyloglucan and rhamnogalacturonan was investigated by similar methods. It appears that it can be broken using a combination of arabinosidase and endoarabinanase. One graduate student, two undergraduate students, one post doctoral fellow and one mentoring professor were involved in performing the experiments. Some of the results were disseminated at the DOE's Physical Biosciences Contractors meeting. PARTICIPANTS: Andrew Mort, PI. Purvi Patel, graduate student, Desra Kiely and Lance Pate, undergraduate students, and Xiangmei Wu, post doctoral fellow. TARGET AUDIENCES: TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
IMPACT: 2007/10 TO 2008/09 We conclude from co-chromatography in multiple different fragments of enzyme digested apple pectin that xylogalacturonan (XGA) and rhamnogalacturonan (RG) are directly linked together, although we still do not know the exact nature of the linkage between them. Beta 1-4 galactans appear to be linked directly to rhamnose of RG. We have obtained evidence that the arabinans linked to rhamnogalacturonan are always linked via a single galactose residue to O-4 of rhamnose residues. We finished work the ability of endopolygalacturonase to digest XGA and published our results. Knowing more about the structure of pectin will help understand the role of pectin in cell growth, its role in food processing, the enzymes that degrade pectin, and even human health benefits of pectin.

Publications

  • Mort A, Zheng Y, Qiu F, Nimtz M, Bell-Eunice G. 2008. Structure of xylogalacturonan fragments from watermelon cell-wall pectin. Endopolygalacturonase can accommodate a xylosyl residue on the galacturonic acid just following the hydrolysis site. Carbohydr. Res. 343: 1212-21.
  • Tian S, Fang X, Wang W, Yu B, Cheng X, et al. 2008. Isolation and identification of oligomers from partial degradation of lime fruit cutin. J. Agric. Food Chem. 56: 10318-25.
  • Zheng Y, Mort A. 2008. Isolation and structural characterization of a novel oligosaccharide from the rhamnogalacturonan of Gossypium hirsutum L. Carbohydr. Res. 343: 1041-9.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Many combinations of sequences of treatments with cloned poysaccharide hydrolases were applied to pectins from cotton, apple, watermelon, and sugar beet with the goal of learning how the different regions within the pectins are linked together. The digestion products were seperated by HPLC and analyzed for their sugar compositions by GLC, and their structures by mass spectrometery and NMR spectroscopy. Two graduate students, one undergraduate student, and one post doctoral fellow and one mentoring professorr were involved in performing the experiments. Some of the results were disseminated at the International Cell Wall Meeting in Copenhagen, Denmark. PARTICIPANTS: Andrew Mort, PI. Purvi Patel and Sayali Saykhedkar, graduate students, Desra Kiely, undergraduate student, and Yun Zheng, post doctoral fellow. TARGET AUDIENCES: The target audience for this project is scientists needing to understand the role of cell walls and their degradation in plant growth, plant disease, and in biofuels production.

Impacts
From the resuts of our experiments we have concluded that xylogalacturonan and rhamnogalacturonan are directly linked togethe, although we still do not know the exact nature of the linkage between them. We have obtained evidence that the arabinans linked to rhamnogalacturonan are always linked via a single galactose residue to O-4 of rhamnose residues. A manuscript describing some of the results has been submitted. Knowing more about the structure of pectin will help understand the role of pectin in cell growth, its role in food processing, the enzymes that degrade pectin, and even human health benefits of pectin.

Publications

  • Naran, R, ML Pierce, and AJ Mort, (2007) Detection and identification of rhamnogalacturonan-lyase activity in intercellular spaces of expanding cotton cotyledons. The Plant Journal 50: 95-107 Zhang, Z, ML Pierce, and AJ Mort. (2007) Changes in homogalacturonans and enzymes degrading them during cotton cotyledon expansion. Phytochemistry 68: 1094-1103.


Progress 10/01/05 to 09/30/06

Outputs
With the availability of a wide variety of cell wall degrading enzymes (cloned into the yeast Pichia pastoris from the fungus Aspergillus nidulans) we have been able to test various combinations and sequences of enzyme treatments to attempt to isolate fragments of pectins representing junctions between different regions of pectins. We obtain about a 10% yield by weight of rhamnogalacturonan (RG) with galactan sidechains and small amounts of homogalacturonan and xylogalacturonan after an exhaustive digestion of citrus pectin with endopolygalacturonase and pectin methyl esterase in the presence of 10 mM oxalate to ensure calcium crosslinking of homogalacturonans is disrupted. Further digestion with rhamnogalacturonase (along with rhamnogalacturonan acetyl esterase) releases small RG oligomers some of which have galactans attached to them leaving larger oligomers with RG, HG,and xylogalacturonan character. The galactan containing oligomers will allow us to determine exactly how galactans are linked to the RG. The behavior of the larger oligomers on ion exchange chromatography clearly shows that there are linkages between the RG and HG, perhaps via xylogalacturonan sections. Xylogalacturonan has not previously been thought to be a part of citrus pectin. We have made progress towards determining exactly how arabinans are linked to RG using two cloned arabinosidases and commercial sugar beet arabinan which contains mostly branched arabinans linked to a small amount of RG. One of the arabinosidases can hydrolyze only the 3-linked arabinose sidechains from the arabinans, and the other can remove all arabinose sidebranches and cleave the backbone to a small segment. After a subsequent treatment with galactosidase and rhamnogalacturonase we expect to be able to isolate oligomers showing the arabinan-RG link. During the analysis of the fragments we generated with the enzyme treatments we have generated a substantial library of 1 and 2 D NMR spectra of oligosaccharides which will help us, and others, fully characterize the structure of pectins.

Impacts
There is an enormous amount of carbohydrate in plant biomass since every plant cell is surrounded by a polysaccharide rich cell wall. In addition to being very abundant cell walls are a major factor controlling cell expansion, and so, plant growth. By learning the structure of plant cell walls we will be able to improve methods for converting biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood. We may also be able to modify plant growth.

Publications

  • Mort, A.J. 2006. Chemistry in the Determination of Cell Wall Structure. In The Science and Lore of the Plant Cell Wall Biosynthesis, Structure and Function (Hayashi, T., ed.: Brown Walker Press. 48-54.
  • Bauer, S., Prasanna, V., Mort, A.J. and Somerville, C.R. 2005. Cloning, expression and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans. Carbohydr. Res., 340:2590-2597.
  • Bauer, S., Vasu, P., Persson, S., Mort, A.J. and Somerville, C.R. 2006. Development and application of a suite of polysaccharide degrading enzymes for analyzing plant cell walls Proc. Nat. Acad. Sci., 103:11417-11422.
  • Wang, X., Huang, Y., Mort, A.J., Zengd, Y., Tauer, C.G. and Cochrane, K.D. 2006. Variation of Taxane Content in Needles of Taxus x media Cultivars with Different Growth Characteristics Z. Naturforsch., 61c:619-624.


Progress 10/01/04 to 09/30/05

Outputs
We have continued to characterize the mode of action of the wide variety of cell wall polysaccharide degrading enzymes from Aspergillus nidulans that we have expressed in the yeast Pichia pastoris Special emphasis was placed on enzymes affecting pectin and xylan. In addition we have applied some of the enzymes for investigation of the mode of linkage between homogalacturonans and rhamnogalacturonan in pectins. We have two xylanases three arabinosidases, and two xylosidases with different specificities towards glucuronoarabinoxylans, arabinoxylans, and pectic arabinanas These will prove invaluable in determining fine structure of these polymers. For analysis of pectins we now have partially characterized two endopolygalacturonases three pectate lyases and two pectin lyases. Each will give a different set of products from homogalacturonans. We also have characterized two exopolygalacturonases and an endoxylogalacaturonase. By sequential treatment of citrus pectin with one of the pectin lyases, ultrafiltration to remove small fragments, digestion with rhamnogalacturonase, and a second ultrafiltration we have obtained a large amount of fairly low molecular weight oligomers (600-5,000 Mwt) representing the backbone of rhamnogalacturonan with various degrees of branching with sidechains which we know include oligogalacturonan fragments because of characteristic signals in their NMR spectra at 6.13 ppm. Such signals indicate non-reducing terminal, unsaturated, methyl esterified galacturonic acid residues left by the action of the pectin lyase. Further purification and characterization of these oligomers will tell us how rhamnogalacturonan nad homogalacturonan is linked together.

Impacts
Since every plant cell is surrounded by a cell wall, here is an enormous amount of biomass in cell walls. In addition to their shear abundance, walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure f plant cell walls, we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • Mort, A.J., Gao, S., Wu, X., and Prade, R. (2005). The need for pure enzymes that degrade cell wall polysaccharides and how to get them. Plant Biosystems 139: 84-87.
  • Van den Bulck, K., Swennen, K., Loosveld, A.-M.A., Courtin, C.M., Brijs, K., Proost, P., Van Damme, J., Van Campenhout, S., Mort, A., Delcour, J.A. (2005). Isolation of cereal arabinogalactan-peptides and structural comparison of their carbohydrate and peptide moieties. Journal of Cereal Science 41: 59-67.
  • Thirumalapura, N.R., Goad, D.W., Mort, A., Morton, R.J., Clarke J., and Malayer, J. (2005). Structural analysis of the O-antigen of Francisella tularensis subspecies tularensis strain OSU 10. Journal of Medical Microbiology 54: 693-695.


Progress 10/01/03 to 09/30/04

Outputs
Taking advantage of the methods that we reported last year for generating cDNAs for cell wall polysaccharide degrading enzymes in Aspergillus nidulans and expressing them in Pichia pastoris, we, in collaboration with the group of Dr. Christopher Somerville, have cloned and expressed over 70 cell wall polysaccharide degrading enzymes. We have done a partial characterization of some of the enzymes including determining their pH and temperature optimum, and mode of action on defined substrates. The mono-specificity of these individually cloned enzymes should make them exceptionally valuable for studying the structure of pectins and other plant cell wall polysaccharides. During the characterization of the modes of action of the enzymes we are learning more about the details of the structure of pectins. By characterizing the fragments produced by hydrolysis with a xylogalacturonase have been able to show that the distribution of xylose sidechains on the homogalacturonan of cotton is quite different from that on the homogalacturonan of watermelon walls. Although most models propose a linear combination of the various regions (homogalacturonan, xylogalacturonan, rhamnogalacturonan, and rhamnogalacturonan II) of pectin to form the whole molecule, it has been proposed recently that homogalacturonans and xylogalacturonans are sidechains on the rhamnogalacturonan. By digesting citrus pectin with a combination of six purified enzymes and fractionation of the resulting fragments by ion exchange chromatography we have isolated a series of fragments that appear to be short fragments of rhamnogalacturonan with stubs of homogalacturonan attached to them. Further characterization of these fragments will allow us to distinguish between the old and new models of pectin.

Impacts
Since every plant cell is surrounded by a cell wall, there is an enormous amount of biomass in cell walls. In addition, to their shear abundance, walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls, we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • Birgisson, Hakon; Hreggvidsson, Gudmundur O.; Fridjonsson, Olafur H.; Mort, Andrew; Kristjansson, Jakob K.; Mattiasson, Bo (2004) Two new thermostable a-L-rhamnosidases from a novel thermophilic bacterium. Enzyme and Microbial Technology 34, 561-571.
  • Ray, Anamika; Macwana, Sunita; Ayoubi, Patricia; Hall Leo, T.; Prade, Rolf; Mort Andrew, J. (2004) Negative subtraction hybridization: an efficient method to isolate large numbers of condition-specific cDNAs BMC Genomics 2004, 5:22
  • William GT Willats, Lesley McCartney, Clare G Steele-King, Susan E Marcus, Andrew J Mort, Miranda Huisman, Gert-Jan van Alebeek, Henk A Schols, Alphons GJ Voragen, Angelique Le Goff, Estelle Bonnin, Jean-Francois Thibault and J Paul Knox. (2004) A xylogalacturonan epitope is specifically associated with plant cell detachment. Planta 218, 673-681.


Progress 10/01/02 to 09/30/03

Outputs
We devoted most of our efforts over the last year to obtaining reliable, plentiful supplies of well-characterized enzymes for degrading plant cell wall polysaccharides. The approach is to clone the cDNAs for the enzymes into the yeast Pichia pastoris for easy expression and purification. The complete genome sequence of the fungus we are using as enzyme source (Aspergillus nidulans) has become available. We, in collaboration with Rolf Prade, developed a computer program which automatically extracted the accession numbers of all fungal cell wall degrading enzymes (and other carbohydrate active enzymes) from the CAZY web site. These were then used to obtain all the amino acid sequences which were then subjected to a Blastp search of the annotated fungal genome. The program automatically extracted the relevant information about hits and tabulated it. Another segment of the program condensed this information into a more manageable table listing all of the potential enzymes coded for in the fungal genome, identifying its amino acid and nucleic acid sequence, how good a match it is to the closest known sequence, and what its function is, most likely. This table makes it easy to survey the range of enzymes the fungus can make and the number of isozymes that may exist for each activity. It appears that the A. nidulans genome contains 386 putative carbohydrate active enzyme genes. Among these at least 35 code for enzymes involved in degradation of pectin. Over the last year we have succeeded in expressing an exopolygalacturonase, an endoxylogalacturonase, and a pectin methylesterase at quite high levels in pichia. Despite reports in the literature of the inability of fungal pectin methylesterases to remove all methyl esters on homogalacturonans the enzyme we produce does seem to be able to remove them all. Having this enzyme should allow us to de-methylesterify pectins without introducing the artifacts, such as beta-elimination and epimerization of galacturonic acid to altruronic acid, often introduced by the more classical alkaline saponification. Xylogalacturonan stretches seem to be closely associated with the region where rhamnogalacturonans, xyloglucan and probably homogalacturonan crosslink. The xylogalacturonase is capable of digesting cotton xylogalacturonan into rather small fragments, mostly of 6 or fewer residues. So far we have completely characterized one abundant fragment by NMR and MS and find it to be GalA linked alpha 1-4 to another GalA which in addition, is substituted beta 1-3 by a xylose residue. Preliminary results with the larger oligomers indicate that the reducing end of most of them is a xylosylated GalA, and the non reducing end is a non-substituted GalA. We hypothesize that the enzyme cleaves xylogalacturonans at xylosylated GalA residues which are followed by a non-substituted GalA. Such an action pattern would make the enzyme invaluable for determining substitution patterns within xylogalacturonans.

Impacts
Since every plant cell is surrounded by a cell wall, there is an enormous amount of biomass in cell walls. In addition, to their shear abundance, walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls, we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • Nicola Wietholter, Barbara Graebner, Manfred Mierau, Andrew J. Mort, Bruno M. Moerschbacher (2003) Differences in the methyl ester distribution of pectins from near-isogenic wheat lines resistant and susceptible to the wheat stem rust fungus. Molecular plant-microbe interactions 16(10), 945-52.


Progress 10/01/01 to 09/30/02

Outputs
A major goal of the project is to determine whether or not homogalacturonan and rhamnogalacturonan regions of pectins exist in the same molecule. We need to know this to be able to make more realistic models of the cell wall and to find out how complicated the biosynthesis of pectin is. We have isolated several small fragments of commercial citrus pectin that appear to represent a covalent junction between the homogalacturonan and rhamnogalacturonan regions of the pectin. Thus was done by digesting a large amount of pectin and separating the products by size and charge, We obtained an oligomer, in about 0.2% yield which appears by mass spectrometry and 2D NMR to have the structure: Rha-GalA- Rha-GalA- Rha-GalA-GalA-GalA. If this is truly its structure this would represent a junction between the two regions. Further 2D NMR analysis and MS/MS sequencing will be done to determine a conclusive structure. We have continued our efforts to isolate a small fragment of pectin containing a crosslink between xyloglucan and rhamnogalacturonan or xylogalacturonan. Unfortunately, even all of the relatively pure enzymes we have available, in combination do not reduce the size of the crosslinked complex to a manageable level. In the near future we expect to have expressed an endoxylogalacturonase and an exopolygalacturonase in Pichia pastoris. These enzymes should cause degradation of the xylogalacturonan portion of the complex, greatly diminishing its size. We have made some progress on the objective of cloning and expressing enzymes helpful for structural studies of cell wall polysaccharides. We have added xylanase, xylosidase, and another arabinanase to our collection. With the release of the entire genome sequences of several filamentous fungi, especially aspergilli, with which my colleague Rolf Prade has a lot of experience, it should become quite easy to design primers for PCR reactions to obtain coding sequences for any known cell wall degrading enzyme. Recently we have been having great success in separating neutral oligosaccharides on a graphitized carbon column using acetonitrile/water gradients. The problem was detection. We have slightly modified a post-column reaction system of Honda et al., in which reducing sugars are reacted with cyanoacetamide to generate a fluorescent product, and this allows us to detect very small quantities of oligosaccharides. We can split a small fraction of the column effluent to the detector and collect the rest for subsequent structural analysis or use as a substrate for characterizing enzymes. . Since the cyanoacetamide reaction is compatible with water miscible organic solvents, we can now use the whole range of chromatography columns that have been used for separating oligosaccharides with high sensitivity detection. The combination of the fluorescence detection with hydrophobic and hydrophilic interaction HPLC column systems will give us a much greater range of possibilities when we are trying to separate complex mixtures of non-charged oligosaccharides for analysis.

Impacts
Since every plant cell is surrounded by a cell wall there is an enormous amount of biomass in cell walls. In addition to their shear abundance walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • Baizid, MA (2002) Induction of pectic enzymes in aspergillus nidulans. M.S. thesis Biochemistry and Molecular Biology. Stillwater, Oklahoma State University: 56
  • Van den Bulck, K, Loosveld, A-MA, Courtin, CM, Proost, P, Van Damme, J, Robben, J, Mort, AJ, Delcour, JA (2002) The amino acid sequence of wheat flour arabinogalactan-peptide is identical to part of grain softness protein gsp-1 and leads to an improved structural model. Cereal Chemistry Cereal Chem. 79(3):329-331
  • Mort, AJ (2002) Interactions between pectins and other polymers. In GB Seymour, JP Knox, eds. Pectins and their manipulation. Blackwell Publishing, Sheffield: 30-51.
  • Mort, AJ and Pierce, ML (2002) Preparation of carbohydrates for analysis by modern chromatography and electrophoresis. In Z El Rassi, ed. Carbohydrate analysis by modern chromatography and electrophoresis. Elsevier, Amsterdam, 3-38.


Progress 10/01/00 to 09/30/01

Outputs
Our goals for this project were to isolate and identify any crosslinks between the pectin and xyloglucan (XG) of cotton suspension culture cell walls and between the cell wall protein, extensin, and the pectin of cotton cell walls. We made a great deal of progress towards the goals but did not succeed in totally identifying either of the crosslinks we sought. This is not very surprising since crosslinks need only account for a very small proportion of the cell walls. Our major advances have been to prove conclusively that the crosslinks we sought do exist and to advance the methodology needed to find and characterize them. Ms. Jun Fu has repeatedly shown that around half of the XG extracted from cotton walls by alkali, after their endopolygalacturonase digestion, is firmly bound to anion exchange columns. This shows that half of the XG has acidic character (most likely conferred by its attachment to pectin which binds strongly to the column). If the material adsorbing to the ion exchange column is then adsorbed to crystalline cellulose, which bind XG but not free pectin, about half of the sugars indicative of pectin bind to the cellulose. The XG and pectin can then be released from the cellulose by strong alkali which disrupts the XG-cellulose interaction. The pectin can be released from the cellulose under very mild conditions by an endoglucanase which digests XG. Early experiments indicated that a commercial endoarabinanase cleaved the crosslink between the XG and pectin. However, by a capillary electrophoresis assay for endoglycanases that we developed, we found contaminating endoglucanase activity in the enzyme preparation. We have introduced a bacterial endoarabinanase into Picia pastoris to induce it to produce the enzyme and secrete it. Since this yeast does not naturally produce cell wall degrading enzymes. This allows us to isolate exclusively arabinase activity. Unfortunately, although the activity is active on fluorescent labeled oligoarabinans, it only cleaves a small proportion of the XG-pectin links. Most of the progress on the characterization of extensin-pectin crosslinks has been in working out the sequences of the proteins. We have isolated two totally distinct cDNAs which correspond to two extensin sequences. Neither one appears to be a full length clone since they do not have AUG codons. We intend to determine the complete sequence of the two extensins by obtaining genomic clones. Northern blots will tell us the approximate length of the full length transcripts. In addition to the extensin clones we repeatedly found clones coding for very proline rich proteins which seem to be fused to a domain which is homologous to an endopolygalacturonase inhibitor protein and a protein kinase. Interestingly, by using the fluorescence based enzyme assay mentioned above, but for endopolygalacturonase(EPG), we have found EPG inhibitor activity in the medium of cotton suspension cultures. We have not yet purified the activity to see if it corresponds to the protein encoded by the cDNA clone.

Impacts
Since every plant cell is surrounded by a cell wall there is an enormous amount of biomass in cell walls. In addition to their shear abundance, walls are a major controling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls we may be able to modify how plants grow.

Publications

  • No publications reported this period


Progress 10/01/99 to 09/30/00

Outputs
Our goal in this project is to determine the complete structure of pectins in cotton suspension culture cell walls and their relationships to other wall polymers. We are attempting to isolate a small fragment of pectin which represents the junction between the rhamnogalacturonan (RG) part of pectin and homogalacturonan (HG) to finally determine whether or not the two are usually covalently linked together. Because we need large amounts of pectin to end up with enough of this fragment we are using commercial citrus pectin. The pectin is degraded by a combination of purified and cloned enzymes and the fragments separated by various types of chromatography. Promising fragments are being characterized by 2D NMR spectroscopy and MALDI mass spectrometry. The structures of the oligomers have not yet been solved. Jun Fu, has isolated, using the RGase, endoglucanase, and EPG, a relatively low molecular weight complex containing a fragment of xyloglucan, some RG, and a heavily xylosylated HG section. We think this represents a crosslink between xyloglucan and pectin, in the region where RG and xylosylated HG join eachother. A similar fragment has been isolated from the walls of cotton cotyledons. At present we do not have pure enzymes capable of degrading this fragment into a small enough size to be fully characterizable. 2D NMR spectra of the fragment show it to contain xylogalacturonan, RG, xyloglucan, and possibly some other oligosaccharide sections. We are hoping to clone enzymes which will allow us to degrade this complex further into small enough pieces for complete characterization. To do this we are differentially screening a cDNA library produced from Aspegillus nidulans grown on a mixture of cell wall polysaccharides as its carbon source compared to the fungus grown on glucose which represses synthesis of cell wall degrading enzymes. Initial trial experiments led to the cloning of a xylanase gene.

Impacts
Since every plant cell is surrounded by a cell wall there is an enormous amount of biomass in cell walls. In addition to their shear abundance walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • No publications reported this period


Progress 10/01/98 to 09/30/99

Outputs
Our goal in this project is to determine the complete structure of pectins in cotton suspension culture cell walls and their relationships to other wall polymers. In the past year we have made progress in three main areas. We have suceeded in expressing a rhamnogalacturonase (RGase) in the yeast, picia. Although the yeast does not produce a large amount of the enzyme, it secretes it into the medium around the yeast, and the enzyme is fused to a his tag so that it is easy to purify. Picia does not use plant cell walls as a carbon source, and so does not produce any cell wall degraing enzymes of its own. Thus we can easily produce RGase in the absence of any contaminating cell wall degrading enzymes. This is extremely difficult to do by purifying cell wall degrading enzymes from plant saprophytic fungi, the normal source for commercial enzymes. RGase degrades the rhamnogalacturonan (RG) backbone of pectins, and so is a perfect compliment to endopolygalacturonase (EPG) which degrades the homogalacturonan (HG) backbone of pectins. RGase is inhibited by sidechains on the RG other than single galactose residues. Single Gal residues are common, but so are more complex ones. Thus, RGase digestion of pectins yields mixtures of small oligomers and larger ones. Our quest to isolate small oligosaccharides containing junction zones between RG and HG is still inhibited by the inability of the RGase to digest the RG with comlex sidechains. We are attempting, in colaboration with Dr. Rolfe Prade in our microbiology department, to mount a large screen for all cell wall degrading enzymes produced by Aspergillus nidulans. We will express these enzymes in the picia. A graduate student in the lab, Ms. Jun Fu, has isolated, using the RGase, endoglucanase, and EPG, a relatively low molecular weight complex containing a fragment of xyloglucan, some RG, and a heavily xylosylated HG section. We think this represents a crosslink between xyloglucan and pectin, in the region where RG and xylosylated HG join eachother. We are hoping that the enzymes isolated from the search mentioned above will allow us to degrade this complex further into small enough pieces for complete characterization. We have evidence for a similar junction zone in commercial citrus pectin, but without the crosslink to the xyloglucan.

Impacts
Since every plant cell is surrounded by a cell wall there is an enormous amount of biomass in cell walls. In addition to their shear abundance walls are a major controlling factor in cell expansion, and hence growth of plants. By learning the structure of plant cell walls we will be able to improve methods to convert biomass into food via ruminants, to fuel via hydrolysis and fermentation, and into materials such as textiles and wood.

Publications

  • Moerschbacher, B. M., Mierau, M., Graessner, B., Noll, U. & Mort, A. J. (1999) Small oligomers of galacturonic acid are endogenous suppressors of disease resistance reactions in wheat leaves. J. Exp. Bot. 50. 605-612.
  • Prade, R. A., Zhan, D., Ayoubi, P. & Mort, A. J. (1999) Pectins, Pectinases and Plant-Microbe Interactions. In Biotechnology and Genetic Engineerining Reviews, Vol. 16 Ed. by S. E. Harding, pp. 361-391. Intercept Limited, Andover


Progress 10/01/97 to 09/30/98

Outputs
During experiments in which we showed that there are few, if any, interruptions of the homogalacturonic acid (HG) regions of pectins with single rhamnose residues we found several oligosaccharides containing a sugar previously unreported in pectins. This was shown by NMR, GLC, and mass spectrometry to be L- altruronic acid. This sugar was probably generated artifactually by alkaline epimerization of the C-5 position of a few of the galacturonic acid residues in the pectin. It could, alternatively be the product of enzymic epimerization such as that used by algae to convert the non-gelling regions of alginate (poly mannuronic acid) into gel forming regions (polyguluronic acid). We have made progress toward finally proving that the rhamnogalacturonan and HG regions of pectins are actually covalently linked together in native pectins. We have isolated small and medium sized fragments which contain pieces of both regions. We plan to characterize these by NMR and mass spectrometry to determine the exact nature of the junction between the two types of region. To help in characterizing oligosaccharides and cell wall degrading enzymes we have been developing capillary electrophoresis methods. We have constructed a laser induced fluorescence detector with autoranging using an intensified CCD camera for the detector. We have also developed the use of scavenger beads to remove excess fluorescence derivatizing reagents. The combination of these two developments allows us to analyze extremely small samples. By labeling the reducing end of a polysaccharide with a fluorescent tag and subsequently degrading the polymer chemically or enzymically we can determine the nature of the region at the reducing end of the polymer. For small polysaccharides and oligosaccharides that have been fluorescently labeled one can see the exact action of glycanases. Last year we reported finding exo- and endopolygalacturonase activity in the intercellular spaces of expanding cotton cotyledons, we now report finding enzyme activity towards the rhamnogalacturonan region of the pectins.

Impacts
(N/A)

Publications

  • Merz, J. M. & Mort, A. J. (1998) A computer controlled variable attenuator for protection and autoranging of a laser induced fluorescence detector for CZE. Electrophoresis 19, 2239-2242.
  • Mort, A. J., Zhan, D. & Rodriguez, V. (1998) Use of scavenger beads to remove excess labeling reagents from CZE samples. Electrophoresis 19, 2129-2132.
  • Zhan, D., Janssen, P. & Mort, A. J. (1998) Scarcity or complete lack of single rhamnose residues interspersed within the homogalacturonan regions of citrus pectin. Carbohydr. Res. 308, 373-380.


Progress 10/01/96 to 09/30/97

Outputs
For many years it has been believed that there are isolated rhamnose residues interspersed between the galacturonic acid residues in the homogalacturonan regions of pectins. These residues were thought to introduce kinks in the otherwise linear structure. We have searched for these rhamnose residues by digesting saponified citrus pectin with a well characterized endopolygalacturonase and looking for the oligosaccharides which would be produced if these isolated residues exist. No such oligosaccharides were found. Instead, we found that the rhamnose was all in regions of pectin containing a GalA-Rha repeating disaccharide backbone. We have developed a capillary electrophoresis method using partial acid hydrolysis, fluorescent labeling, and capillary electrophoresis, to help characterize the structure and size of the GalA-Rha repeating backbone of the pectin and its galactan and arabinan sidechains. By using a fluorescent-labeled fragment of polygalacturonic acid as an in vivo substrate, we have found that there is an excellent correlation between the relative expansion rate of cotton cotyledons and intercellular endopolygalacturonase activity. The level of exopolygalacturonase increases as the expansion rate decreases.

Impacts
(N/A)

Publications

  • YU, L. AND MORT, A. J. (1996) Partial Characterization of Xylogalacturonans from Cell Walls of Ripe Watermelon Fruit: Inhibition of Endopolygalacturonase Activity by Xylosylation. In Pectins and Pectinases, Visser, J., and Voragen, A.G.J., eds.,
  • MIERAU, M., GRAESSNER, B., MORT, A. J. AND MOERSCHBACHER, B. M. (1996) Pectins and Pectinases in Stem Rust Infected Wheat. In Pectins and Pectinases, Visser, J., and Voragen, A.G.J., eds., Elsevier,


Progress 10/01/95 to 09/30/96

Outputs
We have worked out several ways to determine the exact substrate requirements for the enzymes we use to analyze pectins. The endopolygalacturonanse from Aspergillus niger was found to need four adjacent galacturonic acid residues with no methyl ester substituents to be able to act on a pectin. It can digest a pectin in which there are four adjacent, non-esterified GalA residues with the residue at the reducing end of the four substituted with a xylose residue on its 3 position. We have been greatly aided in our investigations of substrate requirements by the use of fluorescent labeling of purified oligosaccharides and separation of them, and their enzyme digestion products, by capillary electrophoresis. Knowing the enzyme specificity has helped us deduce that the rhamnogalacturonan of cotton suspension culture walls is linked directly to a xylogalacturonan, which in turn, is most likely linked to a homogalacturonan. We have found that the pectins of cotton cotyledons differ markedly from those in cultured cells in that they have a much higher overall degree of methylesterification, and that they appear to be somewhat degraded during the expansion of the cotyledons. Investigations using the fluorescent labeled oligosaccharides mentioned above indicate that there are significant levels of pectin degrading enzymes in the intercellular spaces of the cotyledons during their time of greatest expansion.

Impacts
(N/A)

Publications

  • Chen, E.M.W. and Mort, A.J. 1996. Nature of Sites Hydrolyzable by Endopolygalacturonase in Partially Esterified Homogalacturonans. Carbohydrate Polymers 29: 129-136.
  • Mort, A.J. and Chen, E.M.W. 1996. Separation of ANTS-Labeled Oligomers Containing Uronic Acids by Capillary Electrophoresis: Application to Determining the Substrate Specificity of Endopolygalacturonases. Electrophoresis 17: 379-383.
  • Zhang, Z., Pierce, M.L. and Mort, A.J. 1996. Detection and Differentiation of Pectic Enzyme Activity in vitro and in vivo by Capillary Electrophoresis of Products from Fluorescent Labeled Substrate. Electrophoresis 17: 372-378.
  • Nimtz, M., Mort, A.J., Domke, T., Wray, V., Zhang, Y., Qiu, F., Coplin, D. Geider, K. 1996. Structure of Amylovoran, the Capsular Exopolysaccharide from the Fireblight Pathogen Erwinia amylovora. Carbohydrate Res. 287: 59-76.
  • Nimtz, M., Mort, A.J., Domke, T., Wray, V., Zhang, Y., Qiu, F., Coplin, D. and Geider, K. 1996. Structure of Stewartan, the Capsular Exopolysaccharide from the Corn Pathogen Erwinia stewartii. Carbohydrate Res. 288: 189-201.


Progress 10/01/94 to 09/30/95

Outputs
Having gained enough evidence over the last few years to be convinced that thereare covalent linkages between pectin and xyloglucan (XG) and between pectin and the cell wall protein extensin, we have recently been trying to identify the chemical nature of the crosslinks. We have made most progress in trying to isolate the XG-pectin crosslink. The goal is to degrade away most of the XG and pectin, retaining structural integrity of the crosslinking section but in a fragment small enough to characterize by mass spectrometry and/or NMR spectroscopy. We are close to producing small enough crosslinking fragments by using a combination of endoglucanase to degrade the XG, rhamnogalacturonase to degrade the pectin, endoarabinanase to degrade the arabinose sidechains on the pectin, and a chemical degradation using lithium metal in ethylenediamine to degrade pectin not digestible with the rhamnogalacturonase. We have made progress toward showing the nature of the linkage between the rhamnogalacturonan (RG) sections of pectins and some of the "homogalacturonan, (HG)". This HG is resistant to digestion with endopolygalacturonase. We have found a rich source of a similar RG-HG complex in watermelon cell walls and have used it to characterize the enzyme-resistant HG. The cause of the resistance to digestion is substitution on some of the galacturonic acid residues in the pectin with xylose residues (one in four for the watermelon walls and one in three for the cotton walls).

Impacts
(N/A)

Publications


    Progress 10/01/93 to 09/30/94

    Outputs
    Over the last year we have gained enough additional evidence to be convinced that a major fraction of the rhamnogalacturonan of cotton culture cell walls is crosslinked to both the hemicellulosic xyloglucan and the hydroxyproline rich cell wall protein. We have found that we can degrade the rhamnogalacturonan with pure rhamnogalacturonase given to us by a cell wall group in The Netherlands. We can degrade the xyloglucan with a purified commercial endoglucanase, and should be able to degrade the protein with the right combination of proteases. The combination of our ability to solubilize crosslinked cell wall polymers and the activities of these enzymes should allow us to isolate the crosslinking fragments in both cases. Knowing how the various cell wall polymers interact with each other is fundamental to our understanding of cell wall function. While investigating the cell wall crosslinks we developed a post column reaction system for continuous detection of poly-, oligo- or monosaccharides. This detection system should save many man hours for any project dealing with purification of polysaccharides.

    Impacts
    (N/A)

    Publications


      Progress 10/01/92 to 09/30/93

      Outputs
      Over the last year we have gained additional evidence for the existence of covalent linkages between xyloglucans and pectins and between the cell wall protein, extensin, and pectins. Xyloglucan and some rhamnose rich pectin characteristic of rhamnogalacturonan I (RGI) can be solubilized from cotton walls after their digestion with a pure endopolygalacturonase (EPG). A significant proportion of the solubilized xyloglucan can be adsorbed to a Dionex PA1 anion exchange column, indicating that it is covalently linked to negatively charged moities. Digestion of the adsorbed material with cellulose converts most of the glucose and xylose from the complex into non-adsorbed material. By sequential digestion of cell walls with EPG, and cellulose we can solubilize (tilde)70% of the walls without solubilizing the cell wall protein. A subsequent treatment in liquid HF at -73(degree) cleaves most of the arabinofuranosyl residues from the protein making it susceptible to digestion with trypsin. Trypsin solubilized several large slightly glycosylated peptides and some very heavily glycosylated peptides. The highly glycosylated peptides co-fractionate with RGI and cross react in Western blots (as do the less glycosylated ones) with antibody to authentic extensin from tomato. We hope to be able to identify the crosslinks in these complexes by further enzyme degradation with a rhamnogalacturonanase (that we have obtained from the group of A. Voragen in the Netherlands) and endoglucanases or proteases.

      Impacts
      (N/A)

      Publications

      • MERZ, J. M. and MORT, A. J. (1992) The Construction and Use of an Inexpensive Data Collection System for High-Resolution Chromatography. Anal. Biochem. 207, 351-353.
      • WEST, P. R. and MORT, A. J. (1993) "SpectraGraph" and "SpectraSort": Mass Spectral Display and Interpretation Software for the Macintosh. J. Chem. Inf. Comput. Sci. 33, 234-239.
      • MORT, A. J., QIU, F. and MANESS, N. O. (1993) Determination of the Pattern of Methyl Esterification in Pectins. Distribution of Contiguous Non-esterified Residues. Carbohydr. Res. 247, 21-35.
      • PIERCE, M. L., ESSENBERG, M. and MORT, A. J. (1993) A Comparison of the Quantities of Exopolysaccharide Produced by Xanthomonas campestris pv. malvacearum in Susceptible and Resistant Cotton Cotyledons During Early Stages of Infection.


      Progress 10/01/91 to 09/30/92

      Outputs
      By solubilizing the pectins of cotton suspension culture cell walls using eitheranhydrous liquid HF or a pure endopolygalacturonase we have determined that there are four structurally distinct regions. The major portion is almost purely homogalacturonan with a low ((tilde)10%) degree of methyl esterification. This fraction accounts for 20% of the weight of the cell walls. In lower proportion ((tilde)10%) of the wall is a complex region of alternating GalA and Rha residues with galactose or arabinose containing sidechains on about one third of the rhamnose residues. Several years ago we discovered that the galacturonic acid in this region is frequently acetylated. We can now isolate fragments in which we can study the distribution of their acetate esters in relation to the placement of sidechains. Another (tilde)2-3% of the pectin is composed of many different sugars. The remaining portion ((tilde)2-3%) is homogalacturonan but with a high degree of methyl esterification. We have developed a method to determine how the methyl esters are distributed within the homogalacturonans and find that in this highly esterified region that most of the esters are on alternating residues. This finding raises questions of how such a pattern of esterification could be biosynthesized and is in contrast to our finding that commercial fruit pectins with the same degree of esterification are randomly esterified.

      Impacts
      (N/A)

      Publications

      • MANESS, N. O., MIRANDA, E. T. and MORT, A. J. 1991. Recovery of sugar derivatives from 2-aminopyridine labeling mixtures for high performance liquid chromatography using UV or fluorescence detection. J. Chromatog. 587:177-183.
      • LAW, I. J., BRANDT, W. F. and MORT, A. J. 1991. Evidence of differences between mannose-binding lectins from nodules and cotyledons of peanut. Plant Science 79:127-133.


      Progress 10/01/90 to 09/30/91

      Outputs
      In many of the models of cell wall structure which have been proposed it is suggested that there are covalent crosslinks between the major types of polymers. However, the chemical nature of these crosslinks has never been established and their existence has frequently been questioned. We have devised sequential digestions combining enzymic and chemical methods by which we can solubilize fragments of cell walls which contain what appears to be crosslinks between xyloglucan and pectin and between pectin and the major cell wall protein. After homogalacturonans are digested by a purified endopolygalacturonase the xyloglucan and rhamnogalacturonan I (RGI) (a rhamnose rich section of pectin) can be co-solubilized by alkaline extraction. Evidence that these two polymers are covalently linked include co-chromatography on ion exchange chromatogaphy, change in apparent molecular weight of the RGI component upon specific digestion of the xyloglucan by cellulase, and the release of RGI from cell walls by cellulase which does not degrade RGI. After cell walls are digested by both endopolygalacturonase and cellulase all of the cell wall protein remains insoluble. After removal of most of the arabinose residues from the protein by a very mild HF treatment it can be digested with trypsin. This protease co-solubilises peptides and sugars characteristic of RGI. Some of these glycopeptides show a dramatic change in size after complete deglycosylation using liquid HF at 0(degree).

      Impacts
      (N/A)

      Publications


        Progress 10/01/89 to 09/30/90

        Outputs
        We have made considerable progress toward determining the structure of the backbone of the rhamnogalacturonan (RG) of pectin. We, and others, have characterized a repeating disaacharide of galacturonic acid and rhamnose which makes up a large proportion of the region. In previous work, we determined that this disaccharide is acetylated in about 30% of its occurrences. When large fragments of RG were purified, the sugar composition indicated a ratio of 2 galA/rha. Using HF solvolysis, we cleaved the material at many of the rhamnose residues. After this treatment, we obtained a series of fragments representing 1,2,3,4 and 5 of the galA-rha disaccharides, but also obtained oligosaccharides containing a single rhamnose residue along with around 10 of galacturonic acid. Forty percent or more of these galA residues were methyl esterified. We have applied the micro method that we have devised for determining the degree at methyl esterification of pectin to petioles of celery and were able to confirm chemically the indications of distribution revealed by nickel staining (Varner and Taylor) Plant Physiol. 1989.91:31-33.

        Impacts
        (N/A)

        Publications

        • KNIREL, Y.A., VINOGRADOV, E.V. and MORT, A.J. 1989. Application of Anhydrous Hydrogen Fluoride for the Structural Analysis of Polysaccharides. Advances in Carbohydrate Chemistry and Biochemistry 47: 167-202.
        • MANESS, N.O., RYAN, J.D. and MORT, A.J. 1990. Determination of the Degree of Methyl Esterification of Pectins in Small Samples by Selective Reduction of Esterified Galacturonic Acid to Galactose. Anal. Biochem., 185: 3456-352.