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SUMMARY: During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening‐related endo‐POLYGALACTURONASE1 gene. MdPG1‐overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white‐skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel‐associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post‐harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. β‐galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development. [ABSTRACT FROM AUTHOR]

Abstract Background While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta. This study examined the effect of down regulation of PG1 expression in ‘Royal Gala’ apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. Results PG1-suppressed ‘Royal Gala’ apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. Conclusions These findings confirm PG1’s role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.

Abstract Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Metabolites of indoleacetaldehyde (IAAld), indoleethanol (tryptophol, IAAol), tryptamine (TNH2) and indoleacelamide (IAAm) were investigated using 2 system; a bacterial (pea stem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In both system, indoleacetic acid (IAA), IAAol and indolecarboxylic acid (ICA) are produced from IAAld, and IAA and ICA are produced from IAAol (production of IAAld and ICAld not investigated). In the plant system TNIL is converted to IAAld , IAA, IAAol and little ICA and ICAld. In the bacterial system TNH2 is converted to IAA. IAAol, the unknown substance Xb, and many ICA and ICAld. Xb is produced from IAAm, too. A nonacide inhibitor extract from pea stems reduces the IAA gain from IAAld, IAAol and TNH2, and the IAAol and IAAld gain from TNH2; in the bacterial system it increases the IAAol gain from IAAld and the Xb gain from TNH2 and IAAm. Metabolic routes connecting Try, TNH2 and IAA are constructed for the plant tissue and for the epiphytic bacteria, respectively, including 4 sites of inhibitor action. [ABSTRACT FROM AUTHOR]

Metabolites of tryptophan were investigated using 2 systems: a bacterial (pea stem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The plant system produces: indolepyruvic acid (IPyA), indoleacetaldehyde (IAAld), indoleacetic acid (IAA), indoleethanol (trytophol, IAAol), indolecarboxylic acid (ICA), indolecarboxaldehyde (ICAld). Bacteria produce additionally: indoleactic acid (ILA), tryptamine (TNH2) and the unknown Xb and Yb, but IAAld was not detected. A nonacidic inhibitor extract from pea stems decreases the gain of IAA, IPyA, ILA, Yb. It increases the gain of IAAld, IAAol, TNH2, Xb, and (only in the bacterial system) ICA and ICAld. Three sites of inhibitor action are suggested, namely the steps Try → IPyA, TNH2 → IAAld, IAAld → IAA. [ABSTRACT FROM AUTHOR]

Under nonsterile conditions, IAA can be extracted from pea stem sections infiltrated with buffer, IAA, or tryptophan. This IAA has microbial origin, since its occurrence is prevented by antibiotics. All infiltrated IAA disappears in the sections. Under sterile conditions, several inhibitors of IAA oxidase prevent the complete disappearance of infiltrated IAA. Some of them permit, by preventing the disappearance of produced IAA, the formation in vivo of extractable IAA amounts from tryptophan. This IAA production is further increased by pyridoxal (phosphate), and by α-ketoglutarate. [ABSTRACT FROM AUTHOR]

Highlights: [•] NMR was used to study the mobility of polysaccharides in the cell walls of apples. [•] Two Pulse Phase Modulation (TPPM) decoupling significantly improved the resolution. [•] The texture of ‘Scifresh’ apples was markedly firmer than that of ‘Royal Gala’. [•] The arabinans in ‘Scifresh’ were more mobile than those in ‘Royal Gala’. [•] The arabinans were more mobile than the galactans in both cultivars. [ABSTRACT FROM AUTHOR]

Background: Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-beta-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-beta-mannanases to be reinterpreted., Aims: In this review, the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported., Future Outlook: There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.

Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590-600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-beta-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529-538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-beta-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6-11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-beta-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-beta-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.