Similar but different: Determining the mode of action and effects of pectin methylesterases (PME)

master's thesis

• Title (croatian): Slični ali različiti: odreživanje načina djelovanja i utjecaja pektin metilesteraza (PME)
• Author: Tea Martinić-Cezar
• Mentor: Vladimir Mrša (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2021-09-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Pectins are the most abundant components in plant primary cell wall and the most heterogenous cell wall polysaccharides. The most common domain of pectins is homogalacturonan, built on a GalA backbone which can be methylesterfied and acetylated. The structure of homogalacturonan can be modified during plant development due to action of homogalacturonan modifying enzymes. As such, these enzymes play a key role in the maturation and degradation of homogalacturonans and therefore in the control of cell wall rheology. Pectin methylesterases (PMEs) can hydrolyze methyl groups from GalA residues, generating negatively charged groups and creating substrates for other HG-modifying enzymes that can hydrolyze pectins. As PMEs are encoded by a large multigenic family of 66 genes in Arabidopsis, this represents a major challenge for reverse genetic approaches. As an alternative; we chose the exogenous application of recombinant enzyme to study their effects during plant development. During this thesis, five plant (PME-32, PME-2, TT-PME, PME-41 and PME-20) and one fungal (VdPME) PMEs were studied. All proteins were heterogeneously produced in Pichia pastoris as active enzymes and their biochemical properties were compared. For three of them (PME-32, PME-20 and VdPME), their mode of action and their roles in planta were assessed at acidic and alkaline pH. Mode of action was determined using size exclusion chromatography coupled to mass spectrometry, while their roles in planta were assessed through exogenous application of the enzymes and analysis of the phenotypical consequences on dark-grown Arabidopsis thaliana seedlings. This overall showed that the mode of action between PME isoforms differ but appears difficult to predict on the basis of sequences. On the other hand, new approach based on electrostatic potential measurements gives promising results that could greatly facilitate understanding the variability within the PME family.

Abstract (croatian)

Pektini su najzastupljeniji i najheterogeniji polisaharidi biljne stanične stijenke. Homogalakturonan je najčešća domena pektina i izgrađen je od GalA okosnice koja je metilestrificirana i acetilirana. Struktura homogalakturonana može se modificirati tijekom razvoja biljaka djelovanjem homogalakturonan-modificirajućih enzima. Kao takvi, ti enzimi igraju ključnu ulogu u razgradnji homogalakturonana te posljedično kontroliraju reološka svojstva stanične stjenke. Pektin metilesteraze (PME) hidroliziraju metilne skupine homogalakturonana, ostavljajući za sobom negativno nabijene skupine koje postaju supstrati za druge HG-modificirajuće enzime, koji mogu hidrolizirati okosnicu homogalakturonana. Kako su PME kodirani velikom multigeničnom obitelji od 66 gena u Arabidopsis thaliani, to predstavlja veliki izazov za primjenu pristupa reverzine genetike. Kao alternativa; odabrali smo egzogenu primjenu rekombinantnog enzima kako bismo proučili njegove učinke na razvoj biljaka. Tijekom ovog rada proučeno je pet biljnih (PME-32, PME-2, TT-PME, PME-41 i PME-20) i jedan PME porijeklom iz Verticilium dahliae (VdPME). Svi proteini su heterogeno proizvedeni u Pichii pastoris kao aktivni enzimi te su uspoređena njihova biokemijska svojstva. Za tri od njih (PME-32, PME-20 i VdPME), određen je način djelovanja i njihova uloga u biljci pri kiselom i alkalnom pH. Način djelovanja određen je pomoću kromatografije (size exclusion chromatography) povezane sa spektrometrijom masa, dok je njihova uloga u biljci procijenjena egzogenom primjenom enzima i fenotipskom analizom hipokotila Arabidopsis thaliane. Pokazno je da se način djelovanja između izoformi PME razlikuje te je teško predvidljiv na temelju sekvenci. S druge strane, novi pristup temeljen na mjerenjima elektrostatičkog potencijala daje obećavajuće rezultate koji bi mogli uvelike olakšati razumijevanje varijabilnosti unutar PME obitelji.

• Keywords: Arabidopsis thaliana; cell wall; homogalacturonan; oligogalacturonides; pectin; pectin methlyesterase; Verticillium dahliae
• Keywords (english): Arabidopsis thaliana; stanična stijenka; homogalakturonan; oligogalakturonidi; pektin; pektin metilesteraze; Verticillium dahliae
• Language: english
• URN:NBN: urn:nbn:hr:159:519619
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-05-26 11:38:59