Arabinogalactan Proteins Research Articles

Genome-Wide Comparative Analysis of the Fasciclin-like Arabinogalactan Proteins (FLAs) in Salicacea and Identification of Secondary Tissue Development-Related Genes.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix, the sister genus of Populus. In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides. The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis, distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I-IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis, SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow.

Improved Methods for Acetocarmine and Haematoxylin Staining to Visualize Chromosomes in the Filamentous Green Alga Zygnema (Charophyta).

Genome sizes of Zygnema spp. vary greatly, being unknown whether polyploidization occurred. The exact number of chromosomes in this genus is unknown since counting methods established for higher plants cannot be applied to green algae. The massive presence of pectins and arabinogalactan proteins in the cell wall interferes with the uptake of staining solutions; moreover, cell divisions in green algae are not restricted to meristems as in higher plants, which is another limiting factor. Cell divisions occur randomly in the thallus, due to the intercalary growth of algal filaments. Therefore, we increased the number of cell divisions via synchronization by changing the light cycle (10:14 h light/dark). The number of observed mitotic stages peaked at the beginning of the dark cycle. This protocol describes two methods for the visualization of chromosomes in the filamentous green alga Zygnema. Existing protocols were modified, leading to improved acetocarmine and haematoxylin staining methods as investigated by light microscopy. A freeze-shattering approach with liquid nitrogen was applied to increase the accessibility of the haematoxylin dye. These modified protocols allowed reliable chromosome counting in the genus Zygnema. Key features Improved method for chromosome staining in filamentous green algae. Optimized for the Zygnema strains SAG 698-1a (Z. cylindricum), SAG 698-1b (Z. circumcarinatum), and SAG 2419 (Zygnema 'Saalach'). This protocol builds upon the methods of chromosomal staining in green algae developed by Wittmann (1965), Staker (1971), and Fujii and Guerra (1998). Cultivation and synchronization: 14 days; fixation and permeabilization: 24 h; staining: 1 h; image analysis and chromosome number quantification: up to 20 h.

Dynamics of cell wall components and histochemical profile of a rust fungi gall (Basidiomycota: Pucciniales) on Byrsonima variabilis A.Juss. (Malpighiaceae)

Context An obligate biotrophic parasitism with a rust fungus led to gall formation on Byrsonima variabilis. Aims The hypothesis that the host leaf–rust fungi interaction alters the dynamics of plant cell walls and the histochemical profile toward favouring the plant cell-to-fungi cell translocation of metabolites is tested. Methods Gall samples were sectioned and submitted to anatomical, histometric, histochemical, and immunocytochemical techniques to evaluate structural alterations and the detection of primary and secondary metabolites, as well as the epitopes of glycoproteins, pectins, and hemicelluloses. Key results Fungi gall development results in the hypertrophy of the stomatal chamber and the hyperplasia of epidermis and spongy parenchyma. The cell-to-cell translocation of metabolites from plant mesophyll cells toward the rust fungi gall is favoured by the epitopes of homogalacturonans (HGs) and (1 → 5) α-l-arabinans detected in the hyphae passage sites in the pycnial and aecial stages. The arabinogalactan-proteins (AGPs) may favour mycelial nutrition and differentiation, and cell wall adhesion. HGs and arabinans confer porosity to mesophyll cell walls, which favours the traffic of molecules toward the rust fungi gall. Conclusions The unexpected labelling of AGPs, HGs, and arabinans in fungi cell walls is a novelty regarding the plant–fungi interaction. The primary metabolites detected in rust fungi support hyphae growth and spore maturation. Implications The immunolabelling of host plant cell wall components on fungi cell walls indicates the integrative role of some plant cell wall components in the biological process of pathogen colonisation in leaf tissues.

Arabinogalactan protein polysaccharide chains are required for normal biogenesis of plasmodesmata.

Arabinogalactan proteins (AGPs) are a plant-specific family of extracellular proteoglycans characterized by large and complex galactose-rich polysaccharide chains. Functional elucidation of AGPs, however, has been hindered by the high degree of redundancy of AGP genes. To uncover as yet unexplored roles of AGPs in Arabidopsis, a mutant of Hyp O-galactosyltransferase (HPGT), a critical enzyme that catalyzes the common initial step of Hyp-linked arabinogalactan chain biosynthesis, was used. Here we show, using the hpgt1,2,3 triple mutant, that a reduction in functional AGPs leads to a stomatal patterning defect in which two or more stomata are clustered together. This defect is attributed to increased and dysregulated symplastic transport following changes in plasmodesmata structure, such that highly permeable complex branched plasmodesmata with cavities in branching parts increased in the mutant. We also found that the hpgt1,2,3 mutation causes a reduction of cellulose in the cell wall and accumulation of pectin, which controls cell wall porosity. Our results highlight the importance of AGPs in the correct biogenesis of plasmodesmata, possibly acting through the regulation of cell wall properties surrounding the plasmodesmata.

Temporal cell wall changes during cold acclimation and deacclimation and their potential involvement in freezing tolerance and growth.

Plants adapt to freezing stress through cold acclimation, which is induced by nonfreezing low temperatures and accompanied by growth arrest. A later increase in temperature after cold acclimation leads to rapid loss of freezing tolerance and growth resumption, a process called deacclimation. Appropriate regulation of the trade-off between freezing tolerance and growth is necessary for efficient plant development in a changing environment. The cell wall, which mainly consists of polysaccharide polymers, is involved in both freezing tolerance and growth. Still, it is unclear how the balance between freezing tolerance and growth is affected during cold acclimation and deacclimation by the changes in cell wall structure and what role is played by its monosaccharide composition. Therefore, to elucidate the regulatory mechanisms controlling freezing tolerance and growth during cold acclimation and deacclimation, we investigated cell wall changes in detail by sequential fractionation and monosaccharide composition analysis in the model plant Arabidopsis thaliana, for which a plethora of information and mutant lines are available. We found that arabinogalactan proteins and pectic galactan changed in close coordination with changes in freezing tolerance and growth during cold acclimation and deacclimation. On the other hand, arabinan and xyloglucan did not return to nonacclimation levels after deacclimation but stabilized at cold acclimation levels. This indicates that deacclimation does not completely restore cell wall composition to the nonacclimated state but rather changes it to a specific novel composition that is probably a consequence of the loss of freezing tolerance and provides conditions for growth resumption.

Stellate Trichomes in Dionaea muscipula Ellis (Venus Flytrap) Traps, Structure and Functions

The digestive organs of carnivorous plants have external (abaxial) glands and trichomes, which perform various functions. Dionaea muscipula Ellis (the Venus flytrap) is a model carnivorous plant species whose traps are covered by external trichomes. The aim of the study was to fill in the gap regarding the structure of the stellate outer trichomes and their immunocytochemistry and to determine whether these data support the suggestions of other authors about the roles of these trichomes. Light and electron microscopy was used to show the trichomes' structure. Fluorescence microscopy was used to locate the carbohydrate epitopes that are associated with the major cell wall polysaccharides and glycoproteins. The endodermal cells and internal head cells of the trichomes were differentiated as transfer cells, and this supports the idea that stellate trichomes transport solutes and are not only tomentose-like trichomes. Trichome cells differ in the composition of their cell walls, e.g., the cell walls of the internal head cells are enriched with arabinogalactan proteins (AGPs). The cell walls of the outer head cells are poor in both low and highly homogalacturonans (HGs), but the immature trichomes are rich in the pectic polysaccharide (1-4)-β-D-galactan. In the immature traps, young stellate trichomes produce mucilage which may protect the trap surface, and in particular, the trap entrance. However, the role of these trichomes is different when the outer head cells collapse. In the internal head cells, a thick secondary wall cell was deposited, which together with the thick cell walls of the outer head cells played the role of a large apoplastic space. This may suggest that mature stellate trichomes might function as hydathodes, but this should be experimentally proven.

Cas9/gRNA-Mediated Mutations in PtrFLA40 and PtrFLA45 Reveal Redundant Roles in Modulating Wood Cell Size and SCW Synthesis in Poplar

Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar.

Transcriptomic Evidence Reveals Low Gelatinous Layer Biosynthesis in Neolamarckia cadamba after Gravistimulation.

Trees can control their shape and resist gravity by producing tension wood (TW), which is a special wood that results from trees being put under stress. TW is characterized by the presence of a gelatinous layer (G layer) and the differential distribution of cell wall polymers. In this study, we investigated whether or not gravistimulation in N. cadamba resulted in TW with an obvious G layer. The results revealed an absence of an obvious G layer in samples of the upper side of a leaning stem (UW), as well as an accumulation of cellulose and a decrease in lignin content. A negligible change in the content of these polymers was recorded and compared to untreated plant (NW) samples, revealing the presence of a G layer either in much lower concentrations or in a lignified form. A transcriptomic investigation demonstrated a higher expression of cell wall esterase- and hydrolase-related genes in the UW, suggesting an accumulation of noncellulosic sugars in the UW, similar to the spectroscopy results. Furthermore, several G-layer-specific genes were also downregulated, including fasciclin-like arabinogalactan proteins (FLA), beta-galactosidase (BGAL) and chitinase-like proteins (CTL). The gene coexpression network revealed a strong correlation between cell-wall-synthesis-related genes and G-layer-synthesis-specific genes, suggesting their probable antagonistic role during G layer formation. In brief, the G layer in N. cadamba was either synthesized in a very low amount or was lignified during an early stage of growth; further experimental validation is required to understand the exact mechanism and stage of G layer formation in N. cadamba during gravistimulation.

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

BackgroundArabinogalactan 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.ResultsThe results definitely ruled out the influence of AGPs on fungal growth. The immunochemical analyses revealed that AGPs consist mainly of carbohydrate chains composed of β-linked glucuronosyl residues recognized by LM2 and GlcA-β(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.ConclusionsThe 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.

New insights on the expression patterns of specific Arabinogalactan proteins in reproductive tissues of Arabidopsis thaliana.

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using β-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.

Immunolocalization of pectins and arabinogalactan proteins during fruit abscission in olive (Olea europaea L.)

ISHS XXXI International Horticultural Congress (IHC2022): International Symposium on Integrative Approaches to Product Quality in Fruits and Vegetables Immunolocalization of pectins and arabinogalactan proteins during fruit abscission in olive (Olea europaea L.)

Arabinogalactan protein is a molecular and cytological marker of particular stages of the tomato fruit ripening process

Arabinogalactan proteins (AGPs) are constituents of the plant cell walls, which are increasingly being considered as essential molecules in the formation of the extracellular matrix. Given their molecular structure and the advantage of the carbohydrate moiety, the glycosylation level and its modification is assumed as a cause of the functional diversity of AGPs. The aim of current work was to evaluate the molecular features of AGP as a potential molecule involved in the ripening process. For this purpose, tomato fruits were analyzed at specific five stages of ripening using microscopic and molecular tools. The experiment showed that the high content and the occurrence of the AGP molecule with molecular weight around 120 kDa are related to cell wall conditions and the intensity of ongoing glycosylation. At the breaker and/or turning stage, the processes of modification of the sugar moiety and depolymerization begin to prevail. At the red ripe/pink stage, the synthesis process is replaced by the degradation process, which is associated with the disappearance of AGPs with high molecular weights and the appearance of only single sugar residues with very low molecular weights. The occurrence of AGPs with low molecular weight (∼30 kDa) may be used as a marker of the finalization of the ripening process in tomato fruits. All anatomical and morphological alterations in the cell wall confirm the presence of dependencies and connections between components of the cell wall network. We may presume that selected antibodies (JIM13, LM2, LM14) indicate very specific features of the fruit tissue at different stages of ripening; thus, AGP is a molecular and cytological marker of particular stages of the fruit ripening process.

In vivo structural modification of type II arabinogalactans with fungal endo-β-1, 6-galactanase in Arabidopsis.

Arabinogalactan-proteins (AGPs) are mysterious extracellular glycoproteins in plants. Although AGPs are highly conserved, their molecular functions remain obscure. The physiological importance of AGPs has been extensively demonstrated with β-Yariv reagent, which specifically binds to AGPs and upon introduction into cells, causes various deleterious effects including growth inhibition and programmed cell death. However, structural features of AGPs that determine their functions have not been identified with β-Yariv reagent. It is known that AGPs are decorated with large type II arabinogalactans (AGs), which are necessary for their functions. Type II AGs consist of a β-1,3-galactan main chain and β-1,6-galactan side chains with auxiliary sugar residues such as L-arabinose and 4-O-methyl-glucuronic acid. While most side chains are short, long side chains such as β-1,6-galactohexaose (β-1,6-Gal6) also exist in type II AGs. To gain insight into the structures important for AGP functions, in vivo structural modification of β-1,6-galactan side chains was performed in Arabidopsis. We generated transgenic Arabidopsis plants expressing a fungal endo-β-1,6-galactanase, Tv6GAL, that degrades long side chains specifically under the control of dexamethasone (Dex). Two of 6 transgenic lines obtained showed more than 40 times activity of endo-β-1,6-galactanase when treated with Dex. Structural analysis indicated that long side chains such as β-1,6-Gal5 and β-1,6-Gal6 were significantly reduced compared to wild-type plants. Tv6GAL induction caused retarded growth of seedlings, which had a reduced amount of cellulose in cell walls. These results suggest that long β-1,6-galactan side chains are necessary for normal cellulose synthesis and/or deposition as their defect affects cell growth in plants.

Plasma membrane proteomic changes of Arabidopsis DRP1E during cold acclimation in association with the enhancement of freezing tolerance.

The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.

The fasciclin-like arabinogalactan proteins of Camellia oil tree are involved in pollen tube growth

Fasciclin-like arabinogalactan proteins (FLAs) are a class of highly glycosylated glycoproteins that perform crucial functions in plant growth and development. This study was carried out to further explore their roles in pollen tube growth. The results showed that seven members (CoFLA1/2/3/4/7/8/17) of the CoFLAs family were identified by sequence characteristics, and they all possessed the fasciclin 1 (FAS1) domain and H1 and H2 conserved domains. They were all located on the plasma membranes of tobacco epidermal cells, and the GPI-anchor sequences of CoFLA1/2/3/4 determined the membrane localization. In flower tissues, CoFLA2 and CoFLA8 were not expressed in the pollen tube but were expressed in the unpollinated style and ovary; the others were all expressed in the pollen tube. In the pollination-compatible style and ovary, they exhibited different expression patterns. Furthermore, all CoFLAs promoted pollen germination in vitro, while only CoFLA7 significantly promoted pollen tube elongation, and the expression of CoFLA1/3/4/7/17 in pollen tubes was regulated by CoFLA proteins. The ABA and ABA synthetic inhibitor (sodium tungstate, ST) both inhibited pollen tube elongation; however, only ST downregulated the expression of CoFLA1/7/17 and upregulated the expression of CoFLA4. Taken together, these results demonstrate that CoFLAs may be significant in pollen tube growth in C. oleifera and that some CoFLAs may participate in the regulation of ABA signaling.

Unraveling the role of arabinogalactan proteins in promoting embryogenic cultures and enhancing transient gene expression in grapevine

Unraveling the role of arabinogalactan proteins in promoting embryogenic cultures and enhancing transient gene expression in grapevine

Microcystin-LR and cyanobacterial extracts alter the distribution of cell wall matrix components in rice root cells

Cyanobacterial toxins (known as cyanotoxins) disrupt the plant cytoskeleton (i.e. microtubules and F-actin), which is implicated in the regulation of cell wall architecture. Therefore, cyanotoxins are also expected to affect cell wall structure and composition. However, the effects of cyanobacterial toxicity on plant cell wall have not been yet thoroughly studied. Accordingly, the alterations of cell wall matrix after treatments with pure microcystin-LR (MC-LR), or cell extracts of one MC-producing and one non-MC-producing Microcystis strain were studied in differentiated Oryza sativa (rice) root cells. Semi-thin transverse sections of variously treated LR-White-embedded roots underwent immunostaining for various cell wall epitopes, including homogalacturonans (HGs), arabinogalactan-proteins (AGPs), and hemicelluloses. Homogalacturonan and arabinan distribution patterns were altered in the affected roots, while a pectin methylesterase (PME) activity assay revealed that PMEs were also affected. Elevated intracellular Ca2+ levels, along with increased callose and mixed linkage glucans (MLGs) deposition, were also observed after treatment. Xyloglucans appeared unaffected and lignification was not observed. The exact mechanism of cyanobacterial toxicity against the cell wall is to be further investigated.

An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb2+ and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ∼25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development.

Cell wall lignification may be necessary for somatic embryogenesis of areca palm (Areca catechu)

Areca palm (Areca catechu) is the biggest horticultural crop and the fruits are major income for more than 2 million farmers in Hainan province of China. As the increasing of areca nut prices and the threat of lethal yellowing disease, tissue culture seedlings is becoming an alternative choice for elite high yielding and disease resistant planting materials. However, relatively low somatic embryogenesis rate will limit the clonal products, while mechanisms underlying somatic embryogenesis of areca palm remains unclear so far. To this regard, we established the tissue culture system and analyzed transcrptomic changes during transition from embryogenic callus (EC) to somatic embryos (SE). Results showed that peroxidases (PER39 and PER3–1), which were crucial to the last step for lignin biosynthesis in phenylpropanoid biosynthesis pathway, increased significantly (FC ≥ 2.5, p ≤ 0.001). Many hydroxyproline rich glycoproteins (HRGPs), including arabinogalactan proteins (AGPs), extensins (EXTs) and proline rich proteins (PRPs), were also highly expressed (FPKM ≥ 100) in SE. Gene co-expression network analysis showed the correlation of PERs with HRGPs and phytohormone signaling genes, of which, FLA7–1, FLA7–2 (AGPs) and EXT2–1 (EXTs) were involved in cell wall related GO process. Furthermore, mmajority of identified up-regulated transcription factors (TFs) are well-known lignin biosynthesis regulators such as basic helix-loop-helix (bHLH), and homeobox-leucine zipper (HOX) families. These TFs showed high correlations with PERs and HRGPs. Moreover, staining and microscopic observation showed that phenolic acids (precursor of lignin biosynthesis) were preferably accumulated in SE, high accumulation of lignin and AGPs was also observed in vascular of SE, suggesting that cell wall lignification of vascular may be a key step for somatic embryogenesis, and AGPs could also contribute during the process.

Mechanical stress elicits kiwifruit ripening changes in gene expression and metabolic status

Mechanical stress of kiwifruit is of major importance since it is directly related to fruit quality and postharvest losses. However, the role of mechanical stress in kiwifruit postharvest metabolism remains poorly explored. The present work characterizes the impact of mechanical stress, applied as compression forces (147.1 N), in ‘Hayward’ kiwifruit at harvest as well as following short (14 days) or long (3 months) cold (0 ℃) storage. Using several experimental approaches, we documented that the applied compression-imposed stress conditions and induced the fruit ripening process at room temperature (20 ℃). Fluorescent antibody labeling showed that arabinogalactan proteins and xyloglucans were altered due to compression, while gel diffusion assay indicated that pectin methylesterase activity was reduced by the long cold exposure. Intracellular Fluo-3 AM-dependent Ca2+ signals were detected in proximal to the main compressed fruit areas just after mechanical treatment. Several primary metabolites changed (i.e. oxoproline, gamma-aminobutyric acid and malonic acid) in cold-stored pericarp and placenta tissues after 8 h of compression treatment. Transcriptomic data also disclosed numerous genes, which are mainly involved in sugar metabolism, TCA cycle, cell wall, ethylene and hormones signaling, that were affected by mechanical stress in pericarp and especially in placenta. The weighted correlation and network analysis based on the integration of metabolome and transcriptome datasets identified candidate modules involved in compression stress. We also detected potential TFs, including bZIP53, WRKY40 and GATA17, that are involved in compression stress while meta-data analysis enabled identification of several TFs that regulated by both compression and wounding in kiwifruit. This work provides a first characterization of the compression-affected changes in fruit tissues that offers insights into molecular basis of mechanical stress in kiwifruit.