Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development

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Abstract

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1–5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5–14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening “tyrosine-and lysine-rich protein,” suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division.

Introduction

Wounding induces an array of biological responses to protect and heal the damaged area of the tuber. However, the processes associated with these responses are poorly understood, including a lack of knowledge of the expression profiles of genes induced during formation and maturation of the wound periderm (Lulai, 2007b, Ginzberg et al., 2009, Soler et al., 2011). Among the initial responses is development of a suberized closing layer; i.e. suberization of the existing cells at the wound surface, which is closely followed by formation of a meristematic layer of cells referred to as the phellogen or cork cambium. In the initial response, suberin poly(phenolics) (SPP) are the first suberin biopolymers to accumulate on the walls of exposed and neighboring cells followed by suberin poly(aliphatics) and glycerol (Lulai and Corsini, 1998, Bernards, 2002, Lulai, 2007a). The presence of SPP on wound responding cell walls is easily detected by autofluorescence microscopy and is often used to monitor the initiation and progress of these wound-healing processes. Following closing layer development, the wound periderm is formed by the outwardly dividing meristematic phellogen layer that produces numerous layers of phellem cell derivatives that are also suberized and marked by the presence of SPP. The meristematically active phellogen also divides inwardly producing a few layers of phelloderm cell derivatives. The phellem, phellogen and phelloderm are three distinct types of cells that make up the wound periderm. After these distinct cells are in place and an adequate number of phellem cell layers have been produced, the phellogen cell layer becomes non-meristematic and no longer generates phellem cell derivatives; at this point wound periderm formation is complete and wound-periderm maturation may ensue (Lulai and Freeman, 2001, Lulai, 2007a). During wound periderm maturation, the phellem (skin) becomes more tightly attached to the tuber and more resistant to excoriation re-injury (Sabba and Lulai, 2004). Sabba and Lulai (2002) observed that the characteristically thin and fragile cell walls of the phellogen thicken after cessation of meristematic activity. The thickening of the phellogen radial cell walls in wound periderm may provide the necessary strength and robustness to reduce susceptibility to re-injury of the wounds; this is an agriculturally important part of periderm maturation.

The wound-induced formation of the phellogen cell layer is the starting point for cell division and the development of wound periderm. Wound periderm formation involves the periclinal division of phellogen cells, generating organized rectangular files of phellem and phelloderm cells. Hence, the cell cycle will be induced upon injury and is active in phellogen cells during wound periderm formation. Horvath et al. (2006) identified a plant growth regulatory gene StEBP1 encoding an epidermal growth factor binding protein. This regulatory gene is expressed in developing organs, promotes cell proliferation and is required for the expression of CDKB1:1 a form of cyclin-dependent kinase B (Horvath et al., 2006). Zhiponova et al. (2006) showed that CDKB was active in highly proliferating regions and in wounded tissues and proposed that the CDKB participates in a long-term wound response. The CDK regulatory subunit (CKS1At) was shown to be at peak transcript level when cell division was at maximum. Richard et al. (2001) showed that CKS1At was highly expressed in cell suspensions throughout culture. Collectively, these studies suggest that CKS1At and StCDKB could be candidate genes for studying or marking cell cycle aspects in wound periderm development and maturation.

Certain cell wall proteins and pectins may have key roles in strengthening phellogen cell walls after meristematic inactivation, making them more resistance to fracture and the periderm more resistant to excoriation (Sabba and Lulai, 2002, Sabba and Lulai, 2004, Sabba and Lulai, 2005, Kloosterman et al., 2010, Ross et al., 2011). Extensins are a family of hydroxyproline-rich glycoproteins (HRGPs) which are major components in cell walls of dicots and are characterized by a pentapeptide motif of Ser-Hyp4 which is repeated throughout the protein (Showalter, 1993). Extensin accumulation is tissue-specific and expression is elicited by both cellular and environmental factors (Ahn et al., 1998). According to Dey et al. (1997), extensins are associated with important physiological functions, such as secondary cell wall thickening. Also, Deepak et al. (2007) showed that there were higher levels of HRGP transcripts and associated protein in a cultivar of pearl millet resistant to Sclerospora graminicola, the causative agent of pearl millet downy mildew. Another protein involved in cell wall structure is pectin methyl esterase (PME); this gene product can increase the content of unesterified uronic acid residues allowing pectin to have higher Ca2+ binding efficiency, thereby increasing tissue firmness (Ross et al., 2011). Contiguous patterns of de-esterification of homogalacturonan molecules by PME leads to what Siedlecka et al. (2008) described as egg-box structures, which contribute to pectin stabilization and cell wall stiffening. Cannon et al. (2008) proposed that extensin reacts with acidic pectin polymers forming extensin pectate which may be involved in the assembly of new cell walls. These studies indicate that extensin and PME have major roles in the development of a robust cell wall and that they may be linked to or mark wound-healing events.

Bown et al. (1993) screened a cDNA library for extensin-like (Ext-like) cDNAs and found that a separate Ext-like gene existed which is very similar to extensin. In Arabidopsis, a gene was found that did not exactly match the Ser-Pro4 motifs of extensin but shared strong similarities, hence AtEPR1 was categorized as Ext-like (Dubreucq et al., 2000). Furthermore, Bown et al. (1993) found that Ext-like mRNA increased in tuber tissue after wounding. Another cell wall protein, a tyrosine- and lysine-rich protein (TLRP), appears to be involved in the final assembly and architecture of lignified secondary cell walls (Domingo et al., 1994). Domingo et al. (1994) determined that TLRP participates in cross-linking of proteins to cell walls and can be linked to aromatic side chains of lignin. This cell wall protein was also found to be expressed at higher levels in potato tubers within a population that displayed firmer cooked tuber texture (Ross et al., 2011). Collectively, these studies suggest that expression of extensin and TLRP genes may be linked to wound-healing events and wound-periderm maturation.

Despite the commercial importance of wound healing to the potato industry, the technologies and practices used to ensure complete wound healing are decades old, were derived empirically, and relied on coarse physical determination of the degree of healing. The development of improved methods to hasten and ensure proper wound healing will depend on the identification of robust biochemical/molecular markers to gauge the progress of this process under a variety of storage conditions. The goals of this research were two-fold: (1) to determine the effects of wounding on a group of genes encoding proteins of known or presumed importance to the wound healing process, and (2) to determine the utility of these genes as coordinate indicators or molecular markers for wound healing progress and wound periderm maturation between genotypes.

Section snippets

Plant material, storage conditions and wound model system

First generation certified seed minitubers (Solanum tuberosum L., genotypes Russet Burbank (RB) and NDTX4271-5R (ND)) from 2008 and 2009 greenhouse harvests (Valley Tissue Culture, Halstad, MN, USA), were used in this research. The russeted genotype RB and the red skinned genotype ND were selected for comparison because they are genetically diverse and it was previously shown that this russeted genotype and red skinned genotypes differ in periderm maturation (Lulai and Orr, 1993) and wound

Determination of closing layer and phellem cell layer formation via SPP ratings

The SPP rating system was used to assay the accumulation of SPP on outer tangential, radial and inner tangential cell walls of the developing closing layer and associated wound phellem cells. Importantly, this approach also provides: (1) an index for quantification of phellem cell generation (see Supplement Table S1) and (2) determination of the time line for subsequent cessation of wound phellem cell development and initiation of ensuing wound periderm maturation. In this analysis, the SPP

Discussion

A wide range of genes is induced after tuber excoriation and other wounding injuries. These genes encode proteins that play key roles in closing layer and wound periderm development. Closing layer formation followed by generation of the wound periderm provides the necessary barrier to resist pathogen invasion and water loss thereby maintaining tuber quality and reducing loss of fresh weight. The genes examined in this research encode proteins that are either targeted to cell wall polymers,

Acknowledgement

We thank Dr. Larry Campbell for help with statistical analysis.

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