Low oxygen environment effect on the tomato cell wall composition during the fruit ripening process.

BACKGROUND: Oxygen concentration is a key characteristic of the fruit storage environment determining shelf life and fruit quality. The aim of the work was to identify cell wall components that are related to the response to low oxygen conditions in fruit and to determine the effects of such conditions on the ripening process. Tomato (Solanum lycopersicum) fruits at different stages of the ripening process were stored in an anoxic and hypoxic environment, at 0% and 5% oxygen concentrations, respectively. We used comprehensive and comparative methods: from microscopic immunolabelling and estimation of enzymatic activities to detailed molecular approaches. Changes in the composition of extensin, arabinogalactan proteins, rhamnogalacturonan-I, low methyl-esterified homogalacturonan, and high methyl-esterified homogalacturonan were analysed. RESULTS: In-depth molecular analyses showed that low oxygen stress affected the cell wall composition, i.e. changes in protein content, a significantly modified in situ distribution of low methyl-esterified homogalacturonan, appearance of callose deposits, disturbed native activities of ß-1,3-glucanase, endo-ß-1,4-glucanase, and guaiacol peroxidase (GPX), and disruptions in molecular parameters of single cell wall components. Taken together, the data obtained indicate that less significant changes were observed in fruit in the breaker stage than in the case of the red ripe stage. The first symptoms of changes were noted after 24 h, but only after 72 h, more crucial deviations were visible. The 5% oxygen concentration slows down the ripening process and 0% oxygen accelerates the changes taking place during ripening. CONCLUSIONS: The observed molecular reset occurring in tomato cell walls in hypoxic and anoxic conditions seems to be a result of regulatory and protective mechanisms modulating ripening processes.

The modified activity of prolyl 4 hydroxylases reveals the effect of arabinogalactan proteins on changes in the cell wall during the tomato ripening process.

Arabinogalactan proteins (AGPs) are proteoglycans with an unusual molecular structure characterised by the presence of a protein part and carbohydrate chains. Their specific properties at different stages of the fruit ripening programme make AGPs unique markers of this process. An important function of AGPs is to co-form an amorphous extracellular matrix in the cell wall-plasma membrane continuum; thus, changes in the structure of these molecules can determine the presence and distribution of other components. The aim of the current work was to characterise the molecular structure and localisation of AGPs during the fruit ripening process in transgenic lines with silencing and overexpression of SlP4H3 genes (prolyl 4 hydroxylase 3). The objective was accomplished through comprehensive and comparative in situ and ex situ analyses of AGPs from the fruit of transgenic lines and wild-type plants at specific stages of ripening. The experiment showed that changes in prolyl 4 hydroxylases (P4H3) activity affected the content of AGPs and the progress in their modifications in the ongoing ripening process. The analysis of the transgenic lines confirmed the presence of AGPs with high molecular weights (120-60 kDa) at all the examined stages, but a changed pattern of the molecular features of AGPs was found in the last ripening stages, compared to WT. In addition to the AGP molecular changes, morphological modifications of fruit tissue and alterations in the spatio-temporal pattern of AGP distribution at the subcellular level were detected in the transgenic lines with the progression of the ripening process. The work highlights the impact of AGPs and their alterations on the fruit cell wall and changes in AGPs associated with the progression of the ripening process.

A practical guide to in situ and ex situ characterisation of arabinogalactan proteins (AGPs) in fruits.

BACKGROUND: Arabinogalactan proteins (AGPs) are plant cell components found in the extracellular matrix that play crucial roles in fruit growth and development. AGPs demonstrate structural diversity due to the presence of a protein domain and an expanded carbohydrate moiety. Considering their molecular structure, the modification of glycosylation is a primary factor contributing to the functional variety of AGPs. MAIN BODY: Immunocytochemical methods are used for qualitative and quantitative analyses of AGPs in fruit tissues. These include in situ techniques such as immunofluorescence and immunogold labelling for visualising AGP distribution at different cellular levels and ex situ methods such as Western blotting and enzyme-linked immunoenzymatic assays (ELISA) for molecular characterisation and quantitative detection of isolated AGPs. The presented techniques were modified by considering the structure of AGPs and the changes that occur in fruit tissues during the development and ripening processes. These methods are based on antibodies that recognise carbohydrate chains, which are the only commercially available highly AGP-specific tools. These probes recognise AGP epitopes and identify structural modifications and changes in spatio-temporal distribution, shedding light on their functions in fruit. CONCLUSION: This paper provides a concise overview of AGP research methods, emphasising their use in fruit tissue analysis and demonstrating the accessibility gaps in other tools used in such research (e.g. antibodies against protein moieties). It underscores fruit tissue as a valuable source of AGPs and emphasises the potential for future research to understand of AGP synthesis, degradation, and their roles in various physiological processes. Moreover, the application of advanced probes for AGP visualisation is a milestone in obtaining more detailed insights into the localisation and function of these proteins within fruit.

Apple metabolism under oxidative stress affects plant cell wall structure and mechanical properties.

Several studies have shown beneficial effects of short exposure to oxidative stress on stored fruit, such as better preservation, increased firmness, preservation of polyphenolic compounds, and reduced risk of postharvest disorders such as bitter pit and superficial scald in apples. In this study the effect of short-term oxidative stress conditions on the physiology of apple fruit was investigated. Apple fruit of three cultivars were exposed to hypoxic storage conditions of various lengths to induce anaerobiosis. The response of apple fruit to short-term oxidative stress was evaluated by means of cell wall immunolabeling and atomic force microscopy. In addition, the antioxidant capacity and antioxidative activity of apple peels was assessed. Through various techniques, it was shown that short-term oxidative stress conditions promote specific enzymatic activity that induces changes in the cell wall of apple fruit cells. Exposure to short-term stress resulted in the remodeling of cell wall pectic polysaccharides, observed as an increase in the size and complexity of extracted oxalate pectin. Structural changes in the cell wall were followed by an increase in Young's modulus (compressive stiffness of a solid material, expressed as the relationship between stress and axial strain) of the cell wall material. The data presented in this paper show in a novel way how storage under short-term oxidative stress modifies the cell wall of apple fruit at the molecular level.

Review: structure and modifications of arabinogalactan proteins (AGPs).

The aim of this report is to provide general information on the molecular structure and synthesis of arabinogalactan proteins (AGPs) in association to their physiological significance. Assessment of genetic modifications of the activity of enzymes involved in the AGP biosynthesis is an efficient tool to study AGP functions. Thus, P4H (prolyl 4 hydroxylase) mutants, GLCAT (ß-glucuronosyltransferase) mutants, and GH43 (glycoside hydrolase family 43) mutants have been described. We focused on the overview of AGPs modifications observed at the molecular, cellular, and organ levels. Inhibition of the hydroxylation process results in an increase in the intensity of cell divisions and thus, has an impact on root system length and leaf area. In turn, overexpression of P4H genes stimulates the density of root hairs. A mutation in GLCAT genes responsible for the transfer of glucuronic acid to the AGP molecule revealed that the reduction of GlcA in AGP disrupts the substantial assembly of the primary cell wall. Furthermore, silencing of genes encoding GH43, which has the ability to hydrolyze the AGP glycan by removing incorrectly synthesized ß-1,3-galactans, induces changes in the abundance of other cell wall constituents, which finally leads to root growth defects. This information provides insight into AGPs as a crucial players in the structural interactions present in the plant extracellular matrix.

Working towards arabinogalactan proteins (AGPs) from fruit: carbohydrate composition and impact on fungal growth.

BACKGROUND: Arabinogalactan proteins (AGPs) are extracellular matrix constituents involved in plant response to fungal infection. The aim of the current study was to investigate the antifungal effect of AGPs ex situ and to determine the structural features of AGPs that may have an influence on this activity. The features of AGPs isolated from fruit were investigated with molecular tools based on specific monoclonal antibodies recognizing carbohydrate AGP epitopes. The Antifungal (well-diffusion) Susceptibility Test and the Agar Invasion Test were used to assess the impact of AGPs on Penicillium notatum culture. RESULTS: The results definitely ruled out the influence of AGPs on fungal growth. The immunochemical analyses revealed that AGPs consist mainly of carbohydrate chains composed of ß-linked glucuronosyl residues recognized by LM2 and GlcA-ß(1 → 3)-GalA-α(1 → 2) Rha recognized by JIM13, which do not have the same functional properties outside the plant cell in in vitro experimental conditions. CONCLUSIONS: The action of a single cell wall component does not elicit any influence ex situ. The extensive accumulation of glycan chains of AGPs in infected tissue as a result of a complex mechanism occurring in the cell wall emphasizes the importance of dependencies between particular components of the extracellular matrix in response to fungal attack.

Effect of Low Temperature on Changes in AGP Distribution during Development of Bellis perennis Ovules and Anthers.

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.

The role of arabinogalactan proteins (AGPs) in fruit ripening-a review.

Arabinogalactan proteins (AGPs) are proteoglycans challenging researchers for decades. However, despite the extremely interesting polydispersity of their structure and essential application potential, studies of AGPs in fruit are limited, and only a few groups deal with this scientific subject. Here, we summarise the results of pioneering studies on AGPs in fruit tissue with their structure, specific localization pattern, stress factors influencing their presence, and a focus on recent advances. We discuss the properties of AGPs, i.e., binding calcium ions, ability to aggregate, adhesive nature, and crosslinking with other cell wall components that may also be implicated in fruit metabolism. The aim of this review is an attempt to associate well-known features and properties of AGPs with their putative roles in fruit ripening. The putative physiological significance of AGPs might provide additional targets of regulation for fruit developmental programme. A comprehensive understanding of the AGP expression, structure, and untypical features may give new information for agronomic, horticulture, and renewable biomaterial applications.

Investigations of changes in the arabinogalactan proteins (AGPs) structure, size and composition during the fruit ripening process.

Arabinogalactan proteins (AGPs) are ubiquitous cell wall and plasma membrane components and are characterised by extensive glycosylation and heterogeneity of their carbohydrate and protein units. The aim of the study was to evaluate the structural features of AGPs present in apple fruits at different stages of the ripening process. AGPs were extracted using the Yariv reagent and examined using SDS-PAGE, immunoblotting, FT-IR, and AFM. In situ analysis, immunofluorescence (CLSM) and immunogold-labelling (TEM), were performed. We demonstrated that AGPs were indeed present in apple fruits at the different stages of the ripening process. The changes in the amount (1.52-2.08 mg g-1), diameter (152.73-75.05 nm), molecular mass (50-250 kDa), and distribution in the cell of AGPs demonstrate their variable presence and changeable structure during the ripening process. We propose specific wavenumbers, i.e. 1265 cm-1, 1117 cm-1, and 960 cm-1, which could be assigned to AGPs. The immunofluorescence and immunogold-labelling results indicate that the JIM13 antibody is the most characteristic for AGPs in apple fruits. This study quantitatively demonstrated for the first time that AGP accumulation occurs in ripe fruits, which is supported by the highest AGPs content, the highest molecular mass, and the appearance of a specific distribution pattern at the cellular level.

Properties of Arabinogalactan Proteins (AGPs) in Apple (Malus × Domestica) Fruit at Different Stages of Ripening.

Arabinogalactan proteins (AGPs) are constituents of the cell wall-plasma membrane continuum in fruit tissue. The aim of the study was to characterise AGPs contained in fruit by determination of their chemical structure and morphological properties. The results were obtained from in and ex situ investigations and a comparative analysis of AGPs present in Malus × domestica fruit at different stages of ripening from green fruit through the mature stage to over-ripening during fruit storage. The HPLC and colorimetric methods were used for analyses of the composition of monosaccharides and proteins in AGPs extracted from fruit. We have found that AGPs from fruit mainly consists of carbohydrate chains composed predominantly of arabinose, galactose, glucose, galacturonic acid, and xylose. The protein moiety accounts for 3.15-4.58%, which depends on the various phases of ripening. Taken together, our results show that the structural and morphological properties of AGPs and calcium concentration in AGPs are related to the progress of ripening, which is correlated with proper fruit cell wall assembly. In line with the existing knowledge, our data confirmed the typical carbohydrate composition of AGPs and may be the basis for studies regarding their presumed properties of binding calcium ions.

Immunocytochemical studies on the distribution of arabinogalactan proteins (AGPs) as a response to fungal infection in Malus x domestica fruit.

Arabinogalactan proteins (AGPs) are cell components implicated in plant-microbe interactions. Despite the significance of AGPs in response to stress factors, their distribution during development of fungal disease in fruit is unknown. In our work, in situ analysis of AGP arrangement in fruit inoculated with Penicillium spinulosum during the consecutive days of infection development was carried out. For immunolocalization of AGPs, samples were incubated with JIM13, MAC207, LM2, and LM14 antibodies recognizing the AGP carbohydrate moieties. To analyse cell walls without proper action of AGP, an experiment with ß-glucosyl Yariv reagent specifically binding AGPs was performed. The results showed an increase of signal fluorescence in the fruit after 16 days of fungal disease. Higher amounts of the examined epitopes were observed in the infection-altered sites of the fruit, in close vicinity to a surface filled by fungal spores. The results indicate that the Yariv reagent treatment induced progress of the fungal disease. Changes in the AGP presence during the fungal disease confirmed their involvement in defence against pathogen attack in fruit.

Enzymes and vitamin C as factors influencing the presence of arabinogalactan proteins (AGPs) in Solanum lycopersicum fruit.

Arabinogalactan proteins (AGPs) are ubiquitous components of the amorphous plant extracellular matrix. They are characterized by a high proportion of sugar moieties, heterogeneity of their protein backbone and carbohydrate chains. It is known that AGPs form a complex network with other basic constituents in cell wall thus it may also play a role in softening process of fruit. The use of enzymatic degradation and cell wall polysaccharide directed probes are valid analytical tools for the study of developmental modification of the fruit structure. However, it is unknown whether pectolytic enzymes affect AGPs. Thus, the aim of the current work is to detect AGP epitopes in situ to understand the impact of selected degradation enzymes on various carbohydrate moieties of AGPs. Secondly, there are no data with clarification of the impact of vitamin C on fruit ripening processes at the cellular level; hence, we also focused on the effect of vitamin C on the arrangement of AGPs as important constituents of the polysaccharide-proteoglycan network in the fruit cell wall. The results indicate that the distribution of the examined AGP carbohydrate moieties differs, which are related to changes in tissue architecture. The absence of glycan chains causes disruption in establishment of correlations between cell wall constituents and rearrangement in the cell wall structure. The induced modifications of cell walls are not comparable to alterations occurring in naturally ripening fruit, which allows a conclusion that the synergistic action of a wide variety of factors influences ripening.

Analysis of AGP contribution to the dynamic assembly and mechanical properties of cell wall during pollen tube growth.

Arabinogalactan proteins as cell wall structural proteins are involved in fundamental processes during plant development and growth. The aim of this study was to evaluate AGP function in the distribution of pectin, cellulose and callose along Fragaria x ananassa pollen tube and to associate the cell wall structure with local mechanical properties. We used Yariv reagent which interacts with AGPs and allows the observation of the assembly of cell walls without AGPs performing their function. Cytochemical, immunofluorescence labelling and atomic force microscope have been used to characterize the changes in cell wall structure and stiffness. It was shown that disordering of the structure of AGP present in cell walls affects the localization of cellulose, pectins and the secretion of callose. Changes in cell wall assembly are relevant to pollen tube mechanical properties. The stiffness gradient lengthwise through the axis of the pollen tube has demonstrated a significantly higher Young's modulus of the shank region than the growth zone. It has been revealed that the apex of the pollen tube cultured in the presence of Yariv reagent is stiffer (1.68 MPa) than the corresponding region of the pollen tube grown under control conditions (0.13-0.27 MPa). AGP affects the structure of the cell wall by changing the distribution of other components and the modification of their localization, and hence it plays a significant role in the mechanical properties of the cell wall.

Distribution of arabinogalactan proteins and pectins in the cells of apple (Malus × domestica) fruit during post-harvest storage.

Background and Aims: Changes in the arrangement of cell wall components determine cell wall properties (integrity, stiffness), thereby affecting the macro-scale properties of fruits, which are important for consumers and industry. Arabinogalactan proteins (AGPs) are ubiquitous components of the plant cell, in which they have various functions. Currently, AGPs are considered to be one of the less well-known, enigmatic proteoglycans, a consequence of their heterogeneous structure and unclear mechanism of activity. Methods: An immunocytochemical study was conducted to elucidate the distribution of AGPs and pectic polysaccharides contained in apple (Malus × domestica) fruit during senescence. De-esterified homogalacturonan (LM19), methyl-esterified homogalacturonan (LM20), processed arabinan (LM16) and three AGP epitopes (JIM13, JIM15, MAC207) were identified in the fruit at three stages: fresh fruit, and fruit at 1 and 3 months of post-harvest storage. Key Results: Microscopy revealed spatio-temporal changes in the localization of all examined epitopes. Changes of fruit cell wall assembly and its degradation were confirmed by determination of the galacturonic acid content in the WSP (water soluble pectins), CSP (chelator soluble pectins) and DASP (dilute alkali soluble pectins) fractions. Conclusions: The results revealed dependencies between AGPs, arabinan and homogalacturonan distribution in apple fruit, which are correlated with changes in microstructure during senescence. We propose that AGPs are involved in establishment of the cell wall - plasma membrane continuum.

Structural network of arabinogalactan proteins (AGPs) and pectins in apple fruit during ripening and senescence processes.

The cell wall is an essential framework determining the overall form of the plant cell. Our study was focused on the distribution of arabinogalactan proteins (AGPs), arabinan, and homogalacturonan in fruit cells during ripening and storage with emphasis on quantitative analysis of their presence in particular regions of the cell wall - plasma membrane. The localization of the examined compounds was determined with immunohistochemistry techniques and immunogold labelling. Spatio-temporal colocalization between AGPs epitopes - [ßGlcA(1→3)-αGalA(1→2)Rha] recognized by JIM13 and MAC207 antibodies, and arabinan labelled by the LM16 antibody was detected in the inner cell wall layer, in association with the plasma membrane. The specific arrangement of AGP and arabinan epitopes differentiated them from homogalacturonan epitopes, consisting of GalA residues recognized by LM19 and LM20 antibodies in all the examined fruit maturity stages. The disruption of cell wall - plasma membrane continuum, observed during ripening-associated softening process, was associated with both the substantial decrease of AGPs, pectins content and with remodeling of their arrangement. The results indicate that the textural properties of fruit during growth and postharvest storage, an attribute of fruit quality becoming selection criteria for consumers, depend on the existence of dynamic network organizing polysaccharides and glycoproteins in the extracellular matrix.

Arabinogalactan proteins: Immunolocalization in the developing ovary of a facultative apomict Fragaria x ananassa (Duch.).

BACKGROUND AND AIMS: Arabinogalactan proteins are present in the extracellular matrix and their occurrence is developmentally regulated. The studies were carried out to localise arabinogalactan proteins in ovary cells of Fragaria x ananassa Duchesne (strawberry) during megasporogenesis, megagametogenesis, and formation of the embryo. METHODS: The research included studies of ovary histochemistry and immunofluorescence labelling of AGP epitopes was performed with antibodies JIM13, JIM15 and MAC207. The use of the immunogold labelling method allowed specific detection of AGP epitopes at the subcellular level. KEY RESULTS: The localization of AGPs was studied in the cells of the ovary wall and elements building the developing ovule i.e. the integument, nucellus, archespore, megaspores, embryo sac, and embryo of a facultative apomict Fragaria x ananassa cv. 'Mount Everest'. For the first time the presence of AGP epitopes at the stage of a multicellular archespore was described. The occurrence of AGPs in the functional megaspore walls is related to selection of a megaspore continuing development; during later stages of development, AGPs are also evident markers of the female gametophyte. The intense fluorescence indicates the presence of AGPs in the embryo sac wall as well as in the cytoplasm compartment of the egg apparatus and around the secondary nucleus of the central cell. The localization of AGPs in the ovule of F. x ananassa resembles the distribution of these proteins in amphimictic plants. CONCLUSIONS: Arabinogalactan proteins occur in similar parts of the ovule of amphimictic and apomictic plants. The results confirm the participation of AGPs in reproductive structures as a useful marker during development of female gametophyte.