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<I>Lycopersicon Esculentum</I> University of Connecticut OpenCommons@UConn Honors Scholar Theses Honors Scholar Program May 2005 The olecM ular Characterization of a Predicted Glycosyltransferase from Lycopersicon esculentum Alberto G. Distefano University of Connecticut Follow this and additional works at: https://opencommons.uconn.edu/srhonors_theses Recommended Citation Distefano, Alberto G., "The oM lecular Characterization of a Predicted Glycosyltransferase from Lycopersicon esculentum" (2005). Honors Scholar Theses. 1. https://opencommons.uconn.edu/srhonors_theses/1 1 APPROVAL PAGE HONORS THESIS Molecular characterization of a predicted glycosyltransferase from Lycopersicon esculentum Presented by Alberto Giuseppe Distefano Honors Thesis Advisor ___________________________________________ Wolf -Dieter Reiter Honors Academic Advisor ___________________________________________ Andrew Moiseff Department of Molecular and Cell Biology University of Connecticut 2005 2 Ackn owledgements I would like to thank Dr. Wolf -Dieter Reiter for allowing me to work in his lab and for his assistance with my work and thesis. I would also like to thank Dr. Bruce Link for being my very patient mentor in guiding me in this project, and f or teaching me everything I know about plant cell walls and many lab techniques. I thank all of the lab members for creating a great lab experience for me. Most of all, I would like to thank my parents, brother, and sister for their love and support thro ughout my life, especially during my undergraduate career and the years to come. 3 Abstract The synthesis of the plant cell wall is very complex, and understanding how this process occurs will lead to many benefits for futu re research and industries dependent upon cell walls for their products. The recent discovery of the functions of AtMUR3 and AtGT18 in Arabidopsis thaliana as xyloglucan galactosyltransferases has led to the identification of many more putative glycosyltr ansferases in the Arabidopsis genome. Due to the structural differences between the xyloglucans of Arabidopsis and solanaceous plants, we decided to search for putative arabinosyltransferases in the Solanaceae. Solanaceous xyloglucan is substituted by one to two arabinosyl residues at the second xylose position, and sometimes contains an arabinose at the first xylose position. In contrast, Arabidopsis xyloglucan does not contain arabinose, and is substituted by galactose at the second and third xylose position. Furthermore, the second galactose residue in Arabidopsis xyloglucan is usually fucosylated, a modification not found in solanaceous plants. Searching the database of expressed sequence tags (dbEST), we identified many likely glycosyltransferases in solanaceous plants, including tomato (Lycopersicon esculentum ). AtMUR3 and AtGT18 search queries resulted in the identification of three putative glycosyltransferases in L. esculentum, which were tentatively designated LeGT1 , Le1GT18, and Le2GT18. Bas ed on phylogenetic considerations, Le2GT18 was thought to be a putative arabinosyltransferase. The gene was transformed into atmur3 -3 and atgt18 mutant plants, and the resulting plants will be screened for homozygous plants with the inserted gene. The homozygous T2 plants can then be screened for changes in the composition of their cell walls. Because Le2GT18 is 4 thought to be an arabinosyltransferase, the levels of arabinose may be increased in the xyloglucan fraction of the cell wall. If so, further te sting can be performed to reveal the true function of Le2GT18 . 5 Table of Contents APPROVAL PAGE ........................................................................................................ 1 Acknowledgements ......................................................................................................... 2 Abstract ........................................................................................................................... 3 Table of Contents ............................................................................................................ 5 I. Introduction......................................................................................................... 9 A. The Plant Cell Wall ....................................................................................... 12 1) Functions ...................................................................................................... 12 2) Cellulose ....................................................................................................... 13 3) Hemicellulose ............................................................................................... 14 4) Pectin ............................................................................................................ 17 B. Arabidopsis thaliana ....................................................................................... 19 1) The mur3 and gt18 Mutants of Arabidopsis ................................................. 22 2) Homologues to MUR3 and AtGT18 in Lycopersicon esculentum............... 23 II. Results and Discussion ................................................................................. 27 A. Protein Expression .......................................................................................... 30 B. Putative Function of Le2GT18........................................................................ 31 III. Future Directions .......................................................................................... 32 A. Conclusion .................................................................................................... 33 IV. Materials and Methods.................................................................................. 34 A. Plant Material .................................................................................................. 34 B. DNA Extraction............................................................................................... 34 6 C. DNA Sequencing ............................................................................................. 35 D. Polymerase Chain Reaction (PCR) ................................................................. 35 E. PCR Genotyping of Plants ............................................................................... 38 F. Vectors and Transformation ............................................................................ 40 G. Transformed Mutant and Control Plants ......................................................... 43 H. Protein Expression .......................................................................................... 43 V. Literature Cited .............................................................................................. 45 7 List of Figures Figure 1 : The type I primary cell wall of most flower ing plants. .................................................... 11 Figure 2 : Synthesis of cell wall polysaccharides. ........................................................................ 14 Figure 3 : The typical xyloglucan subunit structure in many dicots, including Arabidopsis thaliana ...... 15 Figure 4 : The xyloglucan structure found in solanaceous plants, including Lycopersicon esculentum .... 16 Figure 5 : Phylogenetic tree of the GT47 family members of Arabidopsis thaliana ............................. 21 Figure 6 : Phylogenetic relationship of Subgroup A in the GT47 family of Arabidopsis with se lected genes from Lycopersicon esculentum ......................................................................................... 22 Figure 7 : Amino acid multiple sequence alignment of Arabidopsis MUR3 and GT18 with putative glycosyltransferases from Lycopersicon esculentum .............................................................. 26 Figure 8 : Removal of internal Nco I sites from Le2GT18 using PCR. ............................................. 29 Figure 9 : Simplified visualization of I PCR protocol used in isolating Le2GT18 . ............................... 39 Figure 10 : PCR temperature profiles used in this experiment. ........................................................ 40 Figure 11 : Vectors used in cloning Le2GT18 .............................................................................. 42 8 List of Tables Table 1 : Primers used in Polymerase Chain Reactions…………………………………………...…….37 9 I. Introduction Plant cell walls play an important part in our daily lives. Paper, lumber, textiles, agricultural items, and many other products are made of cell wall material. Studying cell walls may ultimately lead to increases in the economic productivity of industries that use cell wall materials. Some studies have focused on modifying pec tin cross -linking or cell - cell adhesion to increase the shelf life of fruits and vegetables, enhancing dietary fiber content of cereals, improving yields and quality of fibers, and the relative distribution of carbon to wall biomass for use as biofuels (Mi norsky, 2002). Cellulose is the world’s most abundant renewable resource and a major component of cotton fibers and wood, which makes knowledge of its synthesis desirable to increase economic efficiency. One important topic in applied cell wall research is the digestibility of cell wall material by microorganisms. For instance, cellulose can be enzymatically hydrolyzed to glucose, which can then be fermented to ethanol to yield an excellent source of fuel. Unfortunately, naturally occurring cellulose is very difficult to degrade, so modifications of its structure may improve its usefulness as a renewable
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