An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants.

BACKGROUND: The development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture's induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance-Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed. RESULTS: According to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55. CONCLUSIONS: The amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale's GPRE can be increased.

A time-course analysis of Aspergillus terreus secretomes reveals the importance of pectin-degrading enzymes to increase the digestibility of soybean meal.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

Open questions in plant cell wall synthesis.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support cells while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of model and crop plants have facilitated cataloguing and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall-synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

The Impact of Gold Nanoparticles on Somatic Embryogenesis Using the Example of Arabidopsis thaliana.

Although the influence of nanoparticles (NPs) on developmental processes is better understood, little is known about their impact on somatic embryogenesis (SE). This process involves changes in the direction of cell differentiation. Thus, studying the effect of NPs on SE is essential to reveal their impact on cell fate. This study aimed to examine the influence of gold nanoparticles (Au NPs) with different surface charges on the SE of 35S:BBM Arabidopsis thaliana, with particular emphasis on the spatiotemporal localization of pectic arabinogalactan proteins (AGPs) and extensin epitopes in cells changing the direction of their differentiation. The results show that under the influence of nanoparticles, the explant cells of 35S:BBM Arabidopsis thaliana seedling origin did not enter the path of SE. Bulges and the formation of organ-like structures were observed in these explants, in contrast to the control, where somatic embryos developed. Additionally, spatiotemporal changes in the chemical composition of the cell walls during the culture were observed. Under the influence of Au NPs, the following effects were observed: (1) explant cells did not enter the SE pathway, (2) the impacts of Au NPs with different surface charges on the explants were variable, and (3) the compositions of the analyzed pectic AGPs and extensin epitopes were diverse in the cells with different developmental programs: SE (control) and non-SE (treated with Au NPs).

Determination of the Major By-Products of Citrus hystrix Peel and Their Characteristics in the Context of Utilization in the Industry.

Kaffir lime (Citrus hystrix) is a popular citrus in Southeast Asia. Despite the growing interest in the peel of the fruit, the leaves are the most frequently used part of the fruit. The aim of the study was to determine the main by-products of the peel, such as pectins, minerals, essential oil, and bioactive compounds, and to evaluate the possibility of using them in various branches of industry. In the study of the essential oil obtained by hydrodistillation performed using the TGA chromatography technique (GC-MS), sabinene (31.93%), ß-pinene (26%), and limonene (19%) were selected as the most abundant volatile compounds. Nine microelements (Fe, Zn, Cu, Mn, Co, Ni, Cr, Mo, and V), four macroelements (Mg, Ca, K, and Na), and seven ballast substances (Cd, Hg, Pb, Al, V, Sr, and Pt) were also determined using the microwave plasma-atomic emission spectrometry technique (MP-AES). In the case of microelements, iron 32.72 ± 0.39 mg/kg DW (dry weight) had the highest concentration. In the case of macroelements, the calcium content was 9416 ± 34 mg/kg DW. Optimization of the pectin extraction was also performed by selecting citric acid and obtaining a yield of 7.6-17.6% for acid extraction and 9.9-28.2% for ultrasound-assisted extraction (UAE), depending on the temperature used. The obtained pectins were characterized by the degree of methylation, galacturonic acid content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, and DSC (differential scanning calorimetry) analysis. Among bioactive compounds, the contents of polyphenols (22.63 ± 2.12 mg GAE/g DW), flavonoids (2.72 ± 0.25 mg CE/g DW, vitamin C (2.43 ± 0.19 mg Asc), xantoproteins + carotenes (53.8 ± 4.24 ug), anthocyanins (24.8 ± 1.8 mg CGE/kg DW), and chlorophylls A and B (188.5 ± 8.1, 60.4 ± 3.23 µg/g DW) were evaluated. Antioxidant capacity using (cupric ion-reducing antioxidant capacity) CUPRAC and DPPH assays was also provided with the results of 76.98 ± 8.1, and 12.01 ± 1.02 µmol TE/g DW, respectively.

Contrasting effects of polysaccharide components on the cooking properties of roots, tubers and bananas.

BACKGROUND: Consumer preferences for boiled or fried pieces of roots, tubers and bananas (RTBs) are mainly related to their texture. Different raw and cooked RTBs were physiochemically characterized to determine the effect of biochemical components on their cooking properties. RESULTS: Firmness in boiled sweetpotato increases with sugar and amylose contents but no significant correlation was observed between other physicochemical characteristics and cooking behaviour. Hardness of boiled yam can be predicted by dry matter (DM) and galacturonic acid (GalA) levels. For cassava, no significant correlation was found between textural properties of boiled roots and DM, but amylose and Ca2+ content were correlated with firmness, negatively and positively, respectively. Water absorption of cassava root pieces boiled in calcium chloride solutions was much lower, providing indirect evidence that pectins are involved in determining cooking quality. A highly positive correlation between textural attributes and DM was observed for fried plantain, but no significant correlation was found with GalA, although frying slightly reduced GalA. CONCLUSION: The effect of main components on texture after cooking differs for the various RTBs. The effect of global DM and major components (i.e. starch, amylose) is prominent for yam, plantain and sweetpotato. Pectins also play an important role on the texture of boiled yam and play a prominent role for cassava through interaction with Ca2+ . © 2023 Bill and Melinda Gates Foundation. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment.

In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell-wall-degrading enzymes. Here, we characterize the biochemical properties of the symbiosis-induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance of LbGH28A was confirmed by generating RNA interference (RNAi)-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front. These findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.

Sieve tube structural variation in Austrobaileya scandens and its significance for lianescence.

Lianas combine large leaf areas with slender stems, features that require an efficient vascular system. The only extant member of the Austrobaileyaceae is an endemic twining liana of the tropical Australian forests with well-known xylem hydraulics, but the vascular phloem continuum aboveground remains understudied. Microscopy analysis across leaf vein orders and stems of Austrobaileya scandens revealed a low foliar xylem:phloem ratio, with isodiametric vascular elements along the midrib, but tapered across vein orders. Sieve plate pore radii increased from 0.08 µm in minor veins to 0.12 µm in the petiole, but only to 0.20 µm at the stem base, tens of metres away. In easily bent searcher branches, phloem conduits have pectin-rich walls and simple plates, whereas in twining stems, conduits were connected through highly angled and densely porated sieve plates. The hydraulic resistance of phloem conduits in the twisted and elongated stems of A. scandens is large compared with trees of similar stature; phloem hydraulic resistance decreases from leaves to stems, consistent with the efficient delivery of photoassimilates from sources under Münch predictions. Sink strength of a continuously growing canopy might be stronger than in self-supporting understory plants, favoring resource allocation to aerial organs and the attainment of vertical stature.

Plant Polysaccharide Array for Studying Carbohydrate-Binding Proteins.

The specificity of the most plant carbohydrate-binding proteins (CBP), many of which are known only through bioinformatic analysis of the genome, has either not been studied at all or characterized to a limited extent. The task of deciphering the carbohydrate specificity of the proteins can be solved using glycoarrays composed of many tens or even hundreds of glycans immobilized on a glass surface. Plant carbohydrates are the most significant natural ligands for plant proteins; this work shows that plant polysaccharides without additional modification can be immobilized on the surface, bearing N-hydroxysuccinimide activated carboxyl groups. As a result, an array of 113 well-characterized polysaccharides isolated from various plant cell walls, 23 mono- and oligosaccharides - components of polysaccharides, and glycans - ligands for widely known plant lectins was designed. Upon chemical immobilization of polysaccharides, their functional activity was preserved, which was confirmed by the results of interaction with antibodies and the plant lectin ricin. Using the constructed array, a previously unknown ability of ricin to bind polysaccharides was found, which significantly expands the knowledge of its specificity, and it was also found that a large variety of antibodies to plant polysaccharides are present in human peripheral blood.

Talinum triangulare (Jacq.) Willd. mucilage and pectin in the formulation of ibuprofen microspheres.

BACKGROUND: Mucilage and pectin are both natural polymers with the advantages of availability and biodegradability. Microspheres made from biodegradable polymers can break down naturally after performing their tasks. OBJECTIVES: The study aimed to use mucilage and pectin from the leaves of Talinum triangulare (Jacq.) Willd. as polymer matrices for the formulation of microspheres, with ibuprofen as the model drug. MATERIAL AND METHODS: Both polymers were examined under a microscope and evaluated using measurements of viscosity, density, flow properties, swelling power, elemental analysis, Fourier-transform infrared spectroscopy (FTIR), and the degree of esterification (DE) for pectin. The microspheres were prepared using the ionotropic gelation method and alginate:mucilage/pectin at ratios of 1:1 and 1:2. They were assessed for swellability, drug entrapment effectiveness and drug release profile. RESULTS: The mucilage particles were ovoid while pectin particles were irregularly shaped. Pectin had higher particle, bulk and tapped densities than mucilage, while mucilage had a higher swelling power and a better flow than pectin. Talinum triangulare pectin is a low-methoxyl pectin with a DE of 7.14%. The FTIR spectra showed no interaction between the polymers and ibuprofen. The surface morphology of the microspheres without ibuprofen was smooth, while those with ibuprofen revealed a spongy-like mesh. The swelling power of the microspheres was higher in phosphate buffer with a pH of 7.2 than in distilled water. The entrapment efficiency ranged within 39.57-60.43% w/w, with microspheres containing alginate:mucilage/pectin ratio of 1:1 having higher entrapment efficiency. Microspheres with polymer at a ratio of 1:1 provided a longer release (>2 h), while microspheres with polymer blend of 1:2 provided an immediate release of ibuprofen. CONCLUSIONS: The polymers of T. triangulare could be used as matrices in microsphere formulations.

The exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new ß-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.

Idioblasts and peltate hairs as distribution networks for water absorbed by xerophilous leaves.

Capparis odoratissima is a tree species native to semi-arid environments of South America where low soil water availability coexists with frequent night-time fog. A previous study showed that water applied to leaf surfaces enhanced leaf hydration, photosynthesis and growth, but the mechanisms of foliar water uptake are unknown. Here, we combine detailed anatomical evaluations with water and dye uptake experiments in the laboratory, and use immunolocalization of pectin and arabinogalactan protein epitopes to characterize water uptake pathways in leaves. Abaxially, the leaves of C. odoratissima are covered with peltate hairs, while the adaxial surfaces are glabrous. Both surfaces are able to absorb condensed water, but the abaxial surface has higher rates of water uptake. Thousands of idioblasts per cm2 , a higher density than stomata, connect the adaxial leaf surface and the abaxial peltate hairs, both of which contain hygroscopic substances such as arabinogalactan proteins and pectins. The highly specialized anatomy of the leaves of C odoratissima fulfils the dual function of minimizing water loss when stomata are closed, while maintaining the ability to absorb liquid water. Cell-wall related hygroscopic compounds in the peltate hairs and idioblasts create a network of microchannels that maintain leaf hydration and promote water uptake.

Dynamic changes in cell wall composition of mature sunflower leaves under distinct water regimes affect photosynthesis.

In previous work, we identified that exposure to limited water availability induced changes in cell wall composition of mature Helianthus annuus L. leaves that affected mesophyll conductance to CO2 diffusion (gm). However, it is unclear on which timescale these changes in cell wall composition occurred. Here, we subjected H. annuus to control (i.e. water availability), different levels of short-term water deficit stress (ST), long-term water deficit stress (LT), and long-term water deficit stress followed by gradual recoveries addressed at different timescales (LT-Rec) to evaluate the dynamics of modifications in the main composition of cell wall (cellulose, hemicelluloses, pectins and lignins) affecting photosynthesis. During gradual ST treatments, pectins enhancement was associated with gm decline. However, during LT-Rec, pectins content decreased significantly after only 5 h, while hemicelluloses and lignins amounts changed after 24 h, all being uncoupled from gm. Surprisingly, lignins increased by around 200% compared with control and were related to stomatal conductance to gas diffusion (gs) during LT-Rec. Although we suspect that the accuracy of the protocols to determine cell wall composition should be re-evaluated, we demonstrate for the first time that a highly dynamic cell wall composition turnover differently affects photosynthesis in plants subjected to distinct water regimes.

Chemical composition of cell wall changes during developmental stages of galls on Matayba guianensis (Sapindaceae): perspectives obtained by immunocytochemistry analysis.

The development of plant organs depends on cell division, elongation, structural and chemical changes, and reorganization of cell wall components. As phenotype manipulators, galling insects can manipulate the structure and metabolism of host tissues to build the gall. The gall formation depends on the rearrangement of cell wall components to allow cell growth and elongation, key step for the knowledge regarding gall development, and shape acquisition. Herein, we used an immunocytochemical approach to investigate the chemical composition of the cell wall during the development of galls induced by Bystracoccus mataybae (Eriococcidae) on leaflets of Matayba guianensis (Sapindaceae). Different developmental stages of non-galled leaflets (n = 10) and of leaflet galls (n = 10) were collected from the Cerrado (Brazilian savanna) for anatomical and immunocytochemical analysis. We found that the epitopes of (1 → 4) ß-D-galactans and (1 → 5) α-L-arabinans were evident in the tissues of the young and senescent galls. These epitopes seem to be associated with the mechanical stability maintenance and increased gall porosity. As well, the degree of methyl-esterification of pectins changed from the young to the senescent galls and revealed the conservation of juvenile cell and tissue features even in the senescent galls. The extensins detected in senescent galls seem to support their rigidity and structural reinforcement of these bodies. Our results showed a disruption in the pattern of deposition of leaflet cell wall for the construction of M. guianensis galls, with pectin and protein modulation associated with the change of the developmental gall stages.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

Transformation of Pectins into Non-Ionic or Anionic Surfactants Using a One-Pot and Cascade Mode Process.

The present article describes the one-pot synthesis of double- and single-tailed surfactants by a cascade process that involves the hydrolysis/butanolysis of pectins into butyl galacturonate monosaccharides followed by transesterification/transacetalisation processes with fatty alcohols, and subsequent aqueous basic and acid treatments. The cascade mode allows the depolymerisation to proceed more efficiently, and the purification conditions are optimised to make the production of single-tailed surfactants more manufacturable. These products in a pure form or as mixtures with alkyl glycosides resulting from butanolysis and transglycosylation of pectin-derived hexoses, exhibit attractive surface-tension properties, especially for the n-oleyl ᴅ-galactosiduronic acid products. In addition, a readily biodegradability and an absence of aquatic ecotoxicity are shown for the galacturonic acid derivatives possessing an oleyl alkyl chain at the anomeric position.

Isolation, Chemical Characterization and Antioxidant Activity of Pectic Polysaccharides of Fireweed (Epilobium angustifolium L.).

The aim of this study was to isolate pectins with antioxidant activity from the leaves of Epilobium angustifolium L. Two pectins, EA-4.0 and EA-0.8, with galacturonic acid contents of 88 and 91% were isolated from the leaves of E. angustifolium L. by the treatment of plant raw materials with aqueous hydrochloric acid at pH 4.0 and 0.8, respectively. EA-4.0 and EA-0.8 were found to scavenge the DPPH radical in a concentration-dependent manner at 17-133 µg/mL, whereas commercial apple pectin scavenged at 0.5-2 mg/mL. The antioxidant activity of EA-4.0 was the highest and exceeded the activity of EA-0.8 and a commercial apple pectin by 2 and 39 times (IC50-0.050, 0.109 and 1.961 mg/mL), respectively. Pectins EA-4.0 and EA-0.8 were found to possess superoxide radical scavenging activity, with IC50s equal to 0.27 and 0.97 mg/mL, respectively. Correlation analysis of the composition and activity of 32 polysaccharide fractions obtained by enzyme hydrolysis and anionic exchange chromatography revealed that the antioxidant capacity of fireweed pectins is mainly due to phenolics and is partially associated with xylogalacturonan chains. The data obtained demonstrate that pectic polysaccharides appeared to be bioactive components of fireweed leaves with high antioxidant activity, which depend on pH at their extraction.

PRPs localized to the middle lamellae are required for cortical tissue integrity in Medicago truncatula roots.

KEY MESSAGE: A family of repetitive proline-rich proteins interact with acidic pectins and play distinct roles in legume root cell walls affecting cortical and vascular structure. A proline-rich protein (PRP) family, composed of tandemly repeated Pro-Hyp-Val-X-Lys pentapeptide motifs, is found primarily in the Leguminosae. Four distinct size classes within this family are encoded by seven tightly linked genes: MtPRP1, MtPRP2 and MtPRP3, and four nearly identical MtPRP4 genes. Promoter fusions to ß-glucuronidase showed strong expression in the stele of hairy roots for all 4 PRP genes tested, with additional expression in the cortex for PRP1, PRP2 and PRP4. All except MtPRP4 are strongly expressed in non-tumorous roots, and secreted and ionically bound to root cell walls. These PRPs are absent from root epidermal cell walls, and PRP accumulation is highly localized within the walls of root cortical and vascular tissues. Within xylem tissue, PRPs are deposited in secondary thickenings where it is spatially exclusive to lignin. In newly differentiating xylem, PRPs are deposited in the regularly spaced paired-pits and pit membranes that hydraulically connect neighboring xylem elements. Hairpin-RNA knock-down constructs reducing PRP expression in Medicago truncatula hairy root tumors disrupted cortical and vascular patterning. Immunoblots showed that the knockdown tumors had potentially compensating increases in the non-targeted PRPs, all of which cross-react with the anti-PRP antibodies. However, PRP3 knockdown differed from knockdown of PRP1 and PRP2 in that it greatly reduced viability of hairy root tumors. We hypothesize that repetitive PRPs interact with acidic pectins to form block-copolymer gels that can play distinct roles in legume root cell walls.

Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

Immunodetection of Pectic Epitopes, Arabinogalactan Proteins, and Extensins in Mucilage Cells from the Ovules of Pilosella officinarum Vaill. and Taraxacum officinale Agg. (Asteraceae).

The main aim of this study was to compare the cytological difference between ovular mucilage cells in two Asteraceae species-Pilosella officinarum and Taraxacum officinale-in order to determine whether pectic epitopes, arabinogalactan proteins, or extensins are present. The immunocytochemical technique was used. Both the Taracacum and Pilosella genera have been used recently as models for understanding the mechanisms of apomixis. Knowledge of the presence of signal molecules (pectic epitopes, arabinogalactan proteins, and extensins) can help better understand the developmental processes in these plants during seed growth. The results showed that in Pilosella officinarum, there was an accumulation of pectins in the mucilage, including both weakly and highly esterified pectins, which was in contrast to the mucilage of Taraxacum officinale, which had low amounts of these pectins. However, Taraxacum protoplasts of mucilage cells were rich in weakly methyl-esterified pectins. While the mucilage contained arabinogalactan proteins in both of the studied species, the types of arabinogalactan proteins were different. In both of the studied species, extensins were recorded in the transmitting tissues. Arabinogalactan proteins as well as weakly and highly esterified pectins and extensins occurred in close proximity to calcium oxalate crystals in both Taraxacum and Pilosella cells.