A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsisby Vincenzo Lionetti, Felice Cervone, Giulia De Lorenzo

Phytochemistry

About

Year
2015
DOI
10.1016/j.phytochem.2014.07.025
Subject
Molecular Biology / Horticulture / Plant Science / Biochemistry

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Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast adhesion in plant tissues occurs through the cell wall and primarily monocots (Mohnen, 2008). The backbone of pectin is homogalactuet al., 2012, 2014; Raiola et al., 2011; Wolf et al., 2009). Plant PMEs ockwise) patterns 98; Denes et al., esterification pro) which, t ructures th fen the cell wall (Ridley et al., 2001). The Ca -crosslinked critical for tissue integrity, wall plasticity and cell ad (Derbyshire et al., 2007; Ezaki et al., 2005; Willats et al.,

Because HG and HGA can differentially influence development and defense, plants finely tune HG methylesterification and regulate PME activities through endogenous inhibitors indicated as

PME inhibitors (PMEIs) (Bellincampi et al., 2014; Lionetti et al., 2012; Raiola et al., 2004; Reca et al., 2012). Some mutants, shown to be defective in cell adhesion, have a low level of HGA (Bouton et al., 2002; Neumetzler et al., 2012; Rhee and Somerville, 1998). q This paper forms part of a special issue of Phytochemistry dedicated to the memory and legacy of Professor (Godfrey) Paul Bolwell, MA DSc (Oxon). (19462012), internationally-recognised plant biochemist and Regional Editor of Phytochemistry (2004-2012). He is much missed by his friends. ⇑ Corresponding author. Address: Dipartimento di Biologia e Biotecnologie ‘‘C.

Darwin’’, Edificio di Botanica, ‘‘Sapienza’’ Università di Roma, Piazzale Aldo Moro 5,

Roma 00185, Italy. Tel.: +39 06 49912454; fax: +39 06 49912446.

E-mail address: giulia.delorenzo@uniroma1.it (G. De Lorenzo).

Phytochemistry xxx (2014) xxx–xxx

Contents lists availab

Phytoche journal homepage: www.elseat the level of middle lamella, which is composed mainly of pectin (Jarvis et al., 2003; Knox et al., 1990; Willats et al., 1999). Pectin consists of structurally complex polysaccharides that account for nearly 35% of the primary cell wall of dicots and non-graminaceous monocots and about 2–10% of grasses and other commelinoid can lead to random (non-blockwise) or linear (bl of HG de-methylesterification (Catoire et al., 19 2000; Kim et al., 2005). The blockwise de-methyl duces long stretches of acidic HG (henceforth HGA

Ca++-mediated crosslinks, form rigid ‘‘egg-box’’ st ++http://dx.doi.org/10.1016/j.phytochem.2014.07.025 0031-9422/ 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lionetti, V., et al. A lower content of de-methylesterified homogalacturonan improves enzymatic cell separati isolation of mesophyll protoplasts in Arabidopsis. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.025hrough at stifHGA is hesion 2001).1. Introduction

The integrity of multicellular organisms depends upon the establishment and maintenance of stable cellular connections. Cell ronan (HG), a polymer of a-1,4-linked galacturonic acid residues.

HG is secreted in the cell wall as a highly methylesterified polysaccharide (Mohnen, 2008) and is de-methylesterified during growth and defense by apoplastic pectin methylesterases (PMEs) (LionettiArticle history:

Available online xxxx

This paper forms part of a special issue of

Phytochemistry dedicated to the memory and legacy of Professor (Godfrey) Paul

Bolwell, MA DSc (Oxon). (1946–2012), internationally-recognised plant biochemist and Regional Editor of Phytochemistry (2004–2012). He is much missed by his friends.

Keywords:

Arabidopsis thaliana

Pectin

Homogalacturonan

Pectin methylesterase

Pectin methylesterase inhibitors

Cell adhesion

Enzymatic cell separation

Protoplast isolationisolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of demethylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.  2014 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tA lower content of de-methylesterified h enzymatic cell separation and isolation o

Arabidopsisq

Vincenzo Lionetti, Felice Cervone, Giulia De Lorenzo

Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologieogalacturonan improves mesophyll protoplasts in

Darwin’’, ‘‘Sapienza’’ Università di Roma, Piazzale Aldo Moro 5, Roma 00185, Italy le at ScienceDirect mistry vier .com/locate /phytochemon and 2007), display a better enzymatic saccharification (Francocci et al., 2013; Lionetti et al., 2010). Moreover, analysis of mutants inhibitors have been shown so far to recognize AtPME16 (ATPMEPCRD) and AtPME3, both present in fully expanded leaves of transgenic plants (Lionetti et al., 2007). The very low HGA content observed in PMEI-1 plants with respect to PMEI-2 plants is probably due to a more efficient inhibition by AtPMEI-1 of AtPME16 and

AtPME3 in leaves. Indeed, AtPMEI1 is at least 10-fold more active than AtPMEI-2 against Arabidopsis flower PMEs (Wolf et al., 2003). The moderately low level of HGA in pme3 plants with respect to PMEI-1 plants suggests that, besides PME3, PME16 and possibly other enzymes play a role in HG de-methylesterification.

For isolation of Arabidopsis mesophyll protoplasts, different procedures have been described. The most widely used, named