Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis at the transcriptional and post-translational levels

The ripening of fleshy fruits causes complex biochemical changes during the transformation from the developmental program to the ripening process, which is regulated by hundreds to thousands of genes [1–3]. Ethylene is essential for the initiation and completion of tomato (Solanum lycopersicum L.) fruit ripening [1, 4, 5]. Aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS; EC4.4.1.14) and ACC oxidase (ACO; EC1.3.3.6) are responsible for ethylene biosynthesis, which catalyze the conversion of S-adenosyl-methionine to ACC and ACC to ethylene, respectively [6]. There are many innate elicitors that can induce ethylene production in plants, including abscisic acid (ABA), which can phosphorylate the C-terminus of AtACS6 by activating Arabidopsis thaliana’s calcium-dependent protein kinases AtCDPK4 and AtCDPK11 [7], and auxin, which can inhibit ABA-induced stomatal closure by promoting ethylene production [8]. Additionally, the cell wall degradation products released during the regular fruit ripening process can also induce ethylene production, playing an important role in the complex process of fruit ripening [9, 10].

Oligogalacturonic acids (OGs) are oligomers of alpha-1,4-linked galacturonosyl residues released from the cell wall by the hydrolysis of polygalacturonic acids [11] upon microbial infection [12] and mechanical damage [13], as well as during fruit ripening [14]. As a plant innate elicitor, OGs can induce a series of plant responses [15], including ethylene synthesis [16], the inhibition of auxin action [17, 18], the accumulation of phytoalexins [11, 19] and callose, and the production of reactive oxygen species [20] and nitric oxide [21]. Previous studies demonstrated the endogenous accumulation of pectin-derived oligosaccharides, including OGs, in tomato tissues that were just beginning to ripen and these promoted a short increase of ethylene production in MG tomato pericarp discs [22]. Also, the pectin breakdown products of tomato fruits can be induced by pathogen-related enzyme action [23]. These studies indicate that endogenous pectin-derived oligosaccharides exist and function in the normal course of ripening and disease defense in tomatoes. Early studies suggested that OGs could promote ethylene biosynthesis in tomato fruits or discs, and in pear cell suspensions [9, 10, 24]. A mixture of small-sized OGs elicited ethylene production in tomato plants as a response to wounding [25]. OGs with four to six degrees of polymerization (DP) were shown to be more effective in ethylene promotion through their ability to induce the expression of the LeACO1 gene [16, 25]. Although the OGs’ ability to enhance ethylene production has been shown, the mechanisms behind this capability have not been elaborated and whether the OGs impact the fruit ripening progress remains unclear.

Recent studies found that OGs could mediate cell wall signal transduction and are recognized by wall-associated kinases and kinase-like proteins (WAKs and WAKLs, respectively), which contain an extracellular domain, a transmembrane domain, and a cytoplasmic kinase domain [26]. In Arabidopsis, AtWAK1 interacts with cell wall pectins in a calcium-induced conformation and is an OGs receptor [27, 28], OGs can affect many plant developmental and stress responses by activating the mitogen-activated protein kinases (MAPKs) in a WAK2-dependent manner [29, 30]. Activated MAPKs can regulate a series of phytohormones, including salicylic acid, jasmonic acid, and ethylene, which extensively modulate plant growth, development, and stress/defense responses [31, 32]. Several ACS proteins can be regulated by MAPKs through phosphorylation and dephosphorylation [33, 34]. The phosphorylation of AtACS2 and AtACS6 by MAPK6 led to the accumulation of the ACS protein and elevated levels of cellular ACS activity, promoting ethylene production [35, 36]. Calcium-dependent protein kinases (CDPKs) are also implicated in ACS regulation [34]. In tomato, phosphorylation by MAPK and CDPK are both required to promote LeACS2 stability in the wounded tomato pericarp, and the phosphorylation/dephosphorylation of LeACS2 regulates its turnover upstream of the ubiquitin-26S-proteasome degradation pathway [37, 38].

In this study, we found that OGs could promote the ripening of the tomato cultivar ‘Ailsa Craig’ (AC) fruits at the mature green 1 (MG 1) stage by inducing ethylene biosynthesis. Additionally, the transcriptional levels of LeACS2 and LeACO1 were up-regulated in the presence of OGs. OGs also induce the phosphorylation of LeACS2 at Ser-460. These results demonstrated that OGs induced ethylene biosynthesis at transcriptional and post-translational levels, and then promoted the ripening of tomato fruits. In addition, as a candidate OGs receptor, LeWAKL2 was affected by OGs, which remains to be studied further.