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1.  Wound-induced pectin methylesterases enhance banana (Musa spp. AAA) susceptibility to Fusarium oxysporum f. sp. cubense  
Journal of Experimental Botany  2013;64(8):2219-2229.
Recent studies suggest that plant pectin methylesterases (PMEs) are directly involved in plant defence besides their roles in plant development. However, the molecular mechanisms of PME action on pectins are not well understood. In order to understand how PMEs modify pectins during banana (Musa spp.)–Fusarium interaction, the expression and enzyme activities of PMEs in two banana cultivars, highly resistant or susceptible to Fusarium, were compared with each other. Furthermore, the spatial distribution of PMEs and their effect on pectin methylesterification of 10 individual homogalacturonan (HG) epitopes with different degrees of methylesterification (DMs) were also examined. The results showed that, before pathogen treatment, the resistant cultivar displayed higher PME activity than the susceptible cultivar, corresponding well to the lower level of pectin DM. A significant increase in PME expression and activity and a decrease in pectin DM were observed in the susceptible cultivar but not in the resistant cultivar when plants were wounded, which was necessary for successful infection. With the increase of PME in the wounded susceptible cultivar, the JIM5 antigen (low methyestrified HGs) increased. Forty-eight hours after pathogen infection, the PME activity and expression in the susceptible cultivar were higher than those in the resistant cultivar, while the DM was lower. In conclusion, the resistant and the susceptible cultivars differ significantly in their response to wounding. Increased PMEs and thereafter decreased DMs acompanied by increased low methylesterified HGs in the root vascular cylinder appear to play a key role in determination of banana susceptibility to Fusarium.
PMCID: PMC3654420  PMID: 23580752
Banana (Musa spp.); degree of pectin methylesterification; Fusarium wilt; host–; pathogen interaction; pectin methylesterases; wound induced.
2.  Disorganized distribution of homogalacturonan epitopes in cell walls as one possible mechanism for aluminium-induced root growth inhibition in maize 
Annals of Botany  2009;104(2):235-241.
Background and Aims
Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al.
An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance.
Key Results
In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 µm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in ‘B73’, an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In ‘Nongda3138’, an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 µm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 µm LaCl3 for 24 h, resulting in 40 % inhibition of root growth.
Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.
PMCID: PMC2710910  PMID: 19483201
Al toxicity; cell wall; homogalacturnonan; immunofluorescence; methylesterification; pectin
3.  Are Pectins Involved in Cold Acclimation and De-acclimation of Winter Oil-seed Rape Plants? 
Annals of Botany  2008;101(4):521-530.
Background and Aims
The hypothesis was tested that pectin content and methylation degree participate in regulation of cell wall mechanical properties and in this way may affect tissue growth and freezing resistance over the course of plant cold acclimation and de-acclimation.
Experiments were carried on the leaves of two double-haploid lines of winter oil-seed rape (Brassica napus subsp. oleifera), differing in winter survival and resistance to blackleg fungus (Leptosphaeria maculans).
Key Results
Plant acclimation in the cold (2 °C) brought about retardation of leaf expansion, concomitant with development of freezing resistance. These effects were associated with the increases in leaf tensile stiffness, cell wall and pectin contents, pectin methylesterase (EC 3·1·1·11) activity and the low-methylated pectin content, independently of the genotype studied. However, the cold-induced modifications in the cell wall properties were more pronounced in the leaves of the more pathogen-resistant genotype. De-acclimation promoted leaf expansion and reversed most of the cold-induced effects, with the exception of pectin methylesterase activity.
The results show that the temperature-dependent modifications in pectin content and their methyl esterification degree correlate with changes in tensile strength of a leaf tissue, and in this way affect leaf expansion ability and its resistance to freezing and to fungus pathogens.
PMCID: PMC2710196  PMID: 18222909
Brassica napus subsp; oleifera; cell wall; cold acclimation; de-acclimation; freezing; growth; leaf stiffness; pathogen; pectins; pectin methylesterase
4.  Pectin Methylesterase and Pectin Remodelling Differ in the Fibre Walls of Two Gossypium Species with Very Different Fibre Properties 
PLoS ONE  2013;8(6):e65131.
Pectin, a major component of the primary cell walls of dicot plants, is synthesized in Golgi, secreted into the wall as methylesters and subsequently de-esterified by pectin methylesterase (PME). Pectin remodelling by PMEs is known to be important in regulating cell expansion in plants, but has been poorly studied in cotton. In this study, genome-wide analysis showed that PMEs are a large multi-gene family (81 genes) in diploid cotton (Gossypium raimondii), an expansion over the 66 in Arabidopsis and suggests the evolution of new functions in cotton. Relatively few PME genes are expressed highly in fibres based on EST abundance and the five most abundant in fibres were cloned and sequenced from two cotton species. Their significant sequence differences and their stage-specific expression in fibres within a species suggest sub-specialisation during fibre development. We determined the transcript abundance of the five fibre PMEs, total PME enzyme activity, pectin content and extent of de-methylesterification of the pectin in fibre walls of the two cotton species over the first 25–30 days of fibre growth. There was a higher transcript abundance of fibre-PMEs and a higher total PME enzyme activity in G. barbadense (Gb) than in G. hirsutum (Gh) fibres, particularly during late fibre elongation. Total pectin was high, but de-esterified pectin was low during fibre elongation (5–12 dpa) in both Gh and Gb. De-esterified pectin levels rose thereafter when total PME activity increased and this occurred earlier in Gb fibres resulting in a lower degree of esterification in Gb fibres between 17 and 22 dpa. Gb fibres are finer and longer than those of Gh, so differences in pectin remodelling during the transition to wall thickening may be an important factor in influencing final fibre diameter and length, two key quality attributes of cotton fibres.
PMCID: PMC3673955  PMID: 23755181
5.  Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum, revealed a variability in PME proteolytic maturation 
Plant Signaling & Behavior  2012;7(1):59-61.
Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.1 Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca2+ ions during their intracellular transport.2 The highly methylesterified pectins are then secreted into the apoplasm3 and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca2+ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.4 PMEs belong to a large multigene family. Sixty­six PME-related genes are predicted in the Arabidopsis genome.1 Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.5 In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.
PMCID: PMC3357370  PMID: 22301970
Linum usitatissimum; cell wall; pectin; pectin methylesterase; protein maturation
6.  A Comparative Genome Analysis of PME and PMEI Families Reveals the Evolution of Pectin Metabolism in Plant Cell Walls 
PLoS ONE  2013;8(8):e72082.
Pectins are fundamental polysaccharides in the plant primary cell wall. Pectins are synthesized and secreted to cell walls as highly methyl-esterified polymers and then demethyl-esterified by pectin methylesterases (PMEs), which are spatially regulated by pectin methylesterase inhibitors (PMEIs). Although PME and PMEI genes are pivotal in plant cell wall formation, few studies have focused on the evolutionary patterns of the PME and PMEI gene families. In this study, the gene origin, evolution, and expression diversity of these two families were systematically analyzed using 11 representative species, including algae, bryophytes, lycophytes and flowering land plants. The results show that 1) for the two subfamilies (PME and proPME) of PME, the origin of the PME subfamily is consistent with the appearance of pectins in early charophyte cell walls, 2) Whole genome duplication (WGD) and tandem duplication contribute to the expansion of proPME and PMEI families in land plants, 3) Evidence of selection pressure shows that the proPME and PMEI families have rapidly evolved, particularly the PMEI family in vascular plants, and 4) Comparative expression profile analysis of the two families indicates that the eudicot Arabidopsis and monocot rice have different expression patterns. In addition, the gene structure and sequence analyses show that the origin of the PMEI domain may be derived from the neofunctionalization of the pro domain after WGD. This study will advance the evolutionary understanding of the PME and PMEI families and plant cell wall development.
PMCID: PMC3741192  PMID: 23951288
7.  Metabolomic Analysis and Phenylpropanoid Biosynthesis in Hairy Root Culture of Tartary Buckwheat Cultivars 
PLoS ONE  2013;8(6):e65349.
Buckwheat, Fagopyrum tataricum Gaertn., is an important medicinal plant, which contains several phenolic compounds, including one of the highest content of rutin, a phenolic compound with anti-inflammatory properties. An experiment was conducted to investigate the level of expression of various genes in the phenylpropanoid biosynthetic pathway to analyze in vitro production of anthocyanin and phenolic compounds from hairy root cultures derived from 2 cultivars of tartary buckwheat (Hokkai T8 and T10). A total of 47 metabolites were identified by gas chromatography–time-of-flight mass spectrometry (GC-TOFMS) and subjected to principal component analysis (PCA) in order to fully distinguish between Hokkai T8 and T10 hairy roots. The expression levels of phenylpropanoid biosynthetic pathway genes, through qRT-PCR, showed higher expression for almost all the genes in T10 than T8 hairy root except for FtF3’H-2 and FtFLS-2. Rutin, quercetin, gallic acid, caffeic acid, ferulic acid, 4-hydroxybenzoic acid, and 2 anthocyanin compounds were identified in Hokkai T8 and T10 hairy roots. The concentration of rutin and anthocyanin in Hokkai T10 hairy roots of tartary buckwheat was several-fold higher compared with that obtained from Hokkai T8 hairy root. This study provides useful information on the molecular and physiological dynamic processes that are correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in F. tataricum species.
PMCID: PMC3683005  PMID: 23799007
8.  Ralstonia solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence 
Applied and Environmental Microbiology  1998;64(12):4918-4923.
Ralstonia (Pseudomonas) solanacearum causes bacterial wilt, a serious disease of many crop plants. The pathogen produces several extracellular plant cell wall-degrading enzymes, including polygalacturonases (PGs) and pectin methylesterase (Pme). Pme removes methyl groups from pectin, thereby facilitating subsequent breakdown of this cell wall component by PGs, which are known bacterial wilt virulence factors. R. solanacearum PGs could not degrade 93% methylated pectin unless the substrate was first demethylated by Pme, but as the degree of methylation of the pectin substrate decreased, PG activity increased. Primers derived from a published pme sequence generated an 800-bp DNA probe fragment, which identified Pme-encoding plasmids from a R. solanacearum genomic library. A pme chromosomal mutant had no detectable Pme activity in vitro and no longer grew on 93% methylated pectin as a carbon source. Curiously, the pme mutant, which had no detectable PG activity on highly methylated pectin, was just as virulent as the wild-type strain on tomato, eggplant (aubergine), and tobacco. Since PG activity is required for full virulence, this result suggests that the pectin in these particular hosts may not be highly methylated, or that the breakdown of highly methylated pectin is not a significant factor in the disease process in general. A positive response regulator of PG production called PehR was not required for wild-type Pme production. However, a mutant strain lacking PhcA, which is a global regulator of several virulence genes, produced no detectable Pme activity. Thus, pme expression is directly or indirectly regulated by PhcA but not by PehR.
PMCID: PMC90943  PMID: 9835583
9.  Multiscale investigation of mealiness in apple: an atypical role for a pectin methylesterase during fruit maturation 
BMC Plant Biology  2014;14(1):375.
Apple fruit mealiness is one of the most important textural problems that results from an undesirable ripening process during storage. This phenotype is characterized by textural deterioration described as soft, grainy and dry fruit. Despite several studies, little is known about mealiness development and the associated molecular events. In this study, we integrated phenotypic, microscopic, transcriptomic and biochemical analyses to gain insights into the molecular basis of mealiness development.
Instrumental texture characterization allowed the refinement of the definition of apple mealiness. In parallel, a new and simple quantitative test to assess this phenotype was developed.
Six individuals with contrasting mealiness were selected among a progeny and used to perform a global transcriptome analysis during fruit development and cold storage. Potential candidate genes associated with the initiation of mealiness were identified. Amongst these, the expression profile of an early down-regulated transcript similar to an Arabidopsis thaliana pectin methylesterase gene (AtPME2) matched with mealiness development. In silico analyses of this Malus x domestica PME gene (MdPME2) confirmed its specific pattern compared with all other identified MdPME genes. Protein fusion experiments showed that MdPME2 is secreted into the apoplast in accordance with a possible activity on pectin structure. Further microscopic analysis indicated a progressive loss of cell to cell adhesion in mealy apple fruits. Biochemical analysis revealed specific modifications of pectin residues associated with mealiness, without global changes in the degree of methylesterification of pectins.
These data support the role of PME in cell wall remodelling during apple fruit development and ripening and suggest a local action of these enzymes. Mealiness may partially result from qualitative and spatial variations of pectin microarchitecture rather than quantitative pectin differences, and these changes may occur early in fruit development. The specific MdPME2 gene highlighted in this study could be a good early marker of texture unfavourable trait in apple.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0375-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4310206  PMID: 25551767
Apple; Cell wall; Malus domestica; PME; Fruit texture; Transcriptome
10.  Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance 
Planta  2008;228(1):61-78.
Pectin is one of the main components of the plant cell wall that functions as the primary barrier against pathogens. Among the extracellular pectinolytic enzymes, pectin methylesterase (PME) demethylesterifies pectin, which is secreted into the cell wall in a highly methylesterified form. Here, we isolated and functionally characterized the pepper (Capsicum annuum L.) gene CaPMEI1, which encodes a pectin methylesterase inhibitor protein (PMEI), in pepper leaves infected by Xanthomonascampestris pv. vesicatoria (Xcv). CaPMEI1 transcripts are localized in the xylem of vascular bundles in leaf tissues, and pathogens and abiotic stresses can induce differential expression of this gene. Purified recombinant CaPMEI1 protein not only inhibits PME, but also exhibits antifungal activity against some plant pathogenic fungi. Virus-induced gene silencing of CaPMEI1 in pepper confers enhanced susceptibility to Xcv, accompanied by suppressed expression of some defense-related genes. Transgenic ArabidopsisCaPMEI1-overexpression lines exhibit enhanced resistance to Pseudomonas syringae pv. tomato, mannitol and methyl viologen, but not to the biotrophic pathogen Hyaloperonospora parasitica. Together, these results suggest that CaPMEI1, an antifungal protein, may be involved in basal disease resistance, as well as in drought and oxidative stress tolerance in plants.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-008-0719-z) contains supplementary material, which is available to authorized users.
PMCID: PMC2413075  PMID: 18327607
Pectin methylesterase inhibitor protein; Capsicum annuum; Antifungal activity; Disease resistance; Drought tolerance; Oxidative stress tolerance
11.  Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum) 
PLoS ONE  2013;8(11):e78949.
Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca2+) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.
PMCID: PMC3827314  PMID: 24236073
12.  Cell wall traits as potential resources to improve resistance of durum wheat against Fusarium graminearum 
BMC Plant Biology  2015;15:6.
Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line.
A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession “02-5B-318”, a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat.
Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0369-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4298115  PMID: 25597920
Fusarium Head Blight resistance; Wheat; Pectin methylesterase; Cell wall; Fusarium graminearum
13.  HDG11 upregulates cell-wall-loosening protein genes to promote root elongation in Arabidopsis  
Journal of Experimental Botany  2014;65(15):4285-4295.
EDT1/HGD11 coordinately upregulates gene families of cell-wall-loosening proteins to alter cell-wall extensibility and promote primary root elongation.
The gain-of-function mutant edt1 shows significantly enhanced drought tolerance and a well-developed root system including deeper primary roots and more lateral roots. To explore the molecular mechanisms underlying the improved root system of edt1, we performed transcriptome comparison between the wild-type and edt1 roots. One of the interesting findings from the analysis was that several gene families of cell-wall-loosening proteins were upregulated in the mutant roots, including expansins, extensins, xyloglucan endotransglucosylase/hydrolases (XTHs), pectin-related enzymes, and cellulases. Most of these genes contain HD-binding cis-elements in their promoters predominantly with the TTTAATTT sequence, which can be bound by HDG11 in vitro and in vivo. The coordinated expression of these gene families overlaps fast root elongation. Furthermore, overexpression of AtEXPA5, which was dramatically upregulated in edt1, resulted in longer primary roots because cells were more extended longitudinally. When combined by crossing the AtEXPA5-overexpression lines with one pectin methylesterase inhibitor family protein (PMEI) gene (At5g62360)- or one cellulase (CEL) gene (At2g32990)-overexpression lines, the primary roots of the progeny even exceeded both parents in length. Our results demonstrate that HDG11 directly upregulates cell-wall-loosening protein genes, which is correlated with altered root system architecture, and confirm that cell-wall-loosening proteins play important roles in coordinating cell-wall extensibility with root development. The results of transgene experiments showed that expansin works together with PMEI and CEL to generate synergistic effects on primary root elongation, suggesting that different cell-wall-loosening protein families may function in combination to generate optimal effects on root extensibility.
PMCID: PMC4112634  PMID: 24821957
Cell-wall-loosening protein genes; cellulase; edt1; expansin; HDG11; pectin-related enzymes; XTH.
14.  Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth 
BMC Plant Biology  2013;13:183.
There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation.
‘Scifresh’ (slow softening) and ‘Royal Gala’ (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. ‘Scifresh’ apples showed reduced loss of firmness and greater dry matter accumulation compared with ‘Royal Gala’ during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in ‘Scifresh’ were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of ‘Scifresh’ showed larger, more angular shaped cells than ‘Royal Gala’, with less airspaces and denser tissue. Stronger cell adhesion in ripe ‘Scifresh’ resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in ‘Royal Gala’. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in ‘Scifresh’.
Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.
PMCID: PMC4225529  PMID: 24252512
Apple; Cell adhesion; Cell wall; Fruit firmness; Immunofluorescence labelling; Microstructure; Pectin
15.  Rubus fruticosus (blackberry) use as an herbal medicine 
Pharmacognosy Reviews  2014;8(16):101-104.
Wild grown European blackberry Rubus fruticosus) plants are widespread in different parts of northern countries and have been extensively used in herbal medicine. The result show that European blackberry plants are used for herbal medicinal purpose such as antimicrobial, anticancer, antidysentery, antidiabetic, antidiarrheal, and also good antioxidant. Blackberry plant (R. fruticosus) contains tannins, gallic acid, villosin, and iron; fruit contains vitamin C, niacin (nicotinic acid), pectin, sugars, and anthocyanins and also contains of berries albumin, citric acid, malic acid, and pectin. Some selected physicochemical characteristics such as berry weight, protein, pH, total acidity, soluble solid, reducing sugar, vitamin C, total antioxidant capacity, antimicrobial screening of fruit, leaves, root, and stem of R. fruticosus, and total anthocyanins of four preselected wild grown European blackberry (R. fruticosus) fruits are investigated. Significant differences on most of the chemical content detect among the medicinal use. The highest protein content (2%), the genotypes with the antioxidant activity of standard butylated hydroxyanisole (BHA) studies 85.07%. Different cultivars grown in same location consistently show differences in antioxidant capacity.
PMCID: PMC4127818  PMID: 25125882
Anticancer; antidiabetic; antidysentery; antimicrobial; antioxidant; blackberry; phototherapeutics; R. fruticosus; rosaceae
16.  Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum) 
BMC Genomics  2013;14:742.
Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonans in the cell wall; their activity is regulated in part by pectin methylesterase inhibitors (PMEIs). PME activity may result in either rigidification or loosening of the cell wall, depending on the mode of demethylesterification. The activity of PMEs in the middle lamella is expected to affect intrusive elongation of phloem fibers, and their adhesion to adjacent cells. Length and extractability of phloem fibers are qualities important for their industrial uses in textiles and composites. As only three flax PMEs had been previously described, we were motivated to characterize the PME and PMEI gene families of flax.
We identified 105 putative flax PMEs (LuPMEs) and 95 putative PMEIs (LuPMEIs) within the whole-genome assembly. We found experimental evidence for the transcription of 77/105 LuPMEs and 83/95 LuPMEIs, and surveyed the transcript abundance of these in 12 different tissues and stages of development. Six major monophyletic groups of LuPMEs could be defined based on the inferred relationships of flax genes and their presumed orthologs from other species. We searched the LuPMEs and LuPMEIs for conserved residues previously reported to be important for their tertiary structure and function. In the LuPMEs, the most highly conserved residues were catalytic residues while in the LuPMEIs, cysteines forming disulfude bridges between helices α2 and α3 were most highly conserved. In general, the conservation of critical residues was higher in the genes with evidence of transcript expression than in those for which no expression was detected.
The LuPMEs and LuPMEIs comprise large families with complex patterns of transcript expression and a wide range of physical characteristics. We observed that multiple PMEs and PMEIs are expressed in partially overlapping domains, indicative of several genes acting redundantly during most processes. The potential for functional redundancy was highlighted also by the phylogenetic analyses. We were able to identify a subset of PME and PMEIs that appeared particularly relevant to fiber development, which may provide a basis for the improvement of key traits in industrial feedstocks and a better understanding of the physiological roles of PMEs and PMEIs in general.
PMCID: PMC4008260  PMID: 24168262
Flax; Pectin methylesterase; Fiber; Expression analysis; Phylogenetics; PME; PMEI
17.  Interactive Effects of Al, Ca and Other Cations on Root Elongation of Rice Cultivars Under Low pH 
Annals of Botany  2004;95(2):379-385.
• Background and Aims As with other crop species, Al tolerance in rice (Oryza sativa) is widely different among cultivars, and the mechanism for tolerance is unknown. The Ca2+-displacement hypothesis, that is, Al displaces Ca2+ from critical sites in the root apoplast, was predicted to be the essential mechanism for causing Al toxicity in rice cultivars. If displacement of Ca is an essential cause of Al toxicity in rice, Al toxicity may show the same trend as toxicities of elements such as Sr and Ba that are effective in displacing Ca.
• Methods The interactive effects of Al, Ca, Sr and Ba on root elongation of rice cultivars with different Al tolerances were evaluated in hydroponic culture. Al and Ca accumulation in root tips was also investigated.
• Key Results and Conclusions Not only Al but also Sr and Ba applications inhibited root growth of rice cultivars under low Ca conditions. As expected, rice cultivars more tolerant of Sr and Ba were also tolerant of Al (japonica > indica). Although Mg application did not affect Sr or Ba toxicity, Mg alleviated Al toxicity to the same level as Ca application. In addition, Ca application decreased the Al content in root tips without displacement. These results suggest that Ca does not have a specific, irreplaceable role in Al toxicity, unlike Sr and Ba toxicities. Alleviation of Al toxicity with increasing concentrations of Ca in rice cultivars is due to increased ionic strength, not due to decreased Al activity. The difference in Al tolerance between indica and japonica cultivars disappears under high ionic strength conditions, suggesting that different electrochemical characteristics of root-tip cells are related to the significant difference in Al tolerance under low ionic strength conditions.
PMCID: PMC4246835  PMID: 15546924
Aluminium tolerance; aluminium toxicity; barium toxicity; Ca2+-displacement hypothesis; Oryza sativa L.; root cell walls; strontium toxicity; Triticum aestivum L
18.  Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits 
Journal of Experimental Botany  2013;64(12):3803-3815.
Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.
PMCID: PMC3745733  PMID: 23873994
Cell wall; Fragaria×ananassa; fruit ripening; fruit softening; pectins; polygalacturonase.
19.  Genome-wide transcriptomic and phylogenetic analyses reveal distinct aluminum-tolerance mechanisms in the aluminum-accumulating species buckwheat (Fagopyrum tataricum) 
BMC Plant Biology  2015;15:16.
Similar to common buckwheat (Fagopyrum esculentum), tartary buckwheat (Fagopyrum tataricum) shows a high level of aluminum (Al) tolerance and accumulation. However, the molecular mechanisms for Al detoxification and accumulation are still poorly understood. To begin to elucidate the molecular basis of Al tolerance and accumulation, we used the Illumina high-throughput mRNA sequencing (RNA-seq) technology to conduct a genome-wide transcriptome analysis on both tip and basal segments of the roots exposed to Al.
By using the Trinity method for the de novo assembly and cap3 software to reduce the redundancy and chimeras of the transcripts, we constructed 39,815 transcripts with an average length of 1184 bp, among which 20,605 transcripts were annotated by BLAST searches in the NCBI non-redundant protein database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that expression of genes involved in the defense of cell wall toxicity and oxidative stress was preferentially induced by Al stress. Our RNA-seq data also revealed that organic acid metabolism was unlikely to be a rate-limiting step for the Al-induced secretion of organic acids in buckwheat. We identified two citrate transporter genes that were highly induced by Al and potentially involved in the release of citrate into the xylem. In addition, three of four conserved Al-tolerance genes were found to be duplicated in tartary buckwheat and display diverse expression patterns.
Nearly 40,000 high quality transcript contigs were de novo assembled for tartary buckwheat, providing a reference platform for future research work in this plant species. Our differential expression and phylogenetic analysis revealed novel aspects of Al-tolerant mechanisms in buckwheat.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0395-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4307214  PMID: 25603892
Aluminum tolerance; Al-tolerance genes; Buckwheat; Homolog; Organic acid; Transcriptome
20.  Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes 
Journal of Experimental Botany  2008;59(15):4219-4231.
Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44 000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene α-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode α-copaene synthase. Other potential ‘flavour genes’, identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5′-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.
PMCID: PMC2639024  PMID: 18987392
α-Copaene; flavour; microarray; Solanum tuberosum group Phureja; Solanum tuberosum group Tuberosum; texture; volatile
21.  Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture 
Journal of Experimental Botany  2010;62(1):371-381.
Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.
PMCID: PMC2993920  PMID: 20855456
Cell wall; pectin; pectin methyl esterase; potato; texture; tuber
22.  Histological Analysis of the Maturation of Native and Wound Periderm in Potato (Solanum tuberosum L.) Tuber 
Annals of Botany  2002;90(1):1-10.
Maturation of potato (Solanum tuberosum L.) tuber native and wound periderm and development of resistance to periderm abrasion were investigated utilizing cytological and histochemical techniques. Both native and wound periderm consist of three different tissues: phellem, phellogen and phelloderm. It was previously determined that the phellogen walls of immature native periderm are thin and prone to fracture during harvest, leading to periderm abrasion (excoriation). Phellogen walls thicken and become less susceptible to fracture upon maturation of the periderm, leading to resistance to excoriation. We now demonstrate that phellogen cells of immature wound periderm also have thin radial walls and that wound periderm abrasion is due to fracture of these walls. Maturation of the wound periderm is also associated with an increase in the thickness of the phellogen radial walls. Histological analysis with ruthenium red and hydroxylamine–FeCl2, which stain unesterified and highly methyl‐esterified pectins, respectively, indicates that the phellogen cell walls of native and wound periderm differ significantly regardless of the stage of maturity. Results obtained by staining with ruthenium red and hydroxylamine–FeCl2 imply that phellogen cell walls of immature native periderm contain methyl‐esterified pectin, but are lacking in unesterified (acidic) pectins. Maturation of native periderm is accompanied by an apparent increase in unesterified pectins in the walls of phellogen cells, which may allow for the strengthening of phellogen cell walls via calcium pectate formation. Histological staining of the phellogen walls of wound periderm, on the other hand, implies that these walls are deficient in pectins. Moreover, maturation of wound periderm is not accompanied by an increase in unesterified pectins in these walls. Since peroxidase is known to catalyse the cross‐linking of cell wall polymers, we stained native and wound periderm for the presence of peroxidase utilizing guaiacol as a substrate. Peroxidase staining was strong in the phellogen walls of both immature and mature native periderm and we could not detect any differences in staining between them. Peroxidase staining was weak in the phellogen walls of immature wound periderm and was not detectably different in mature wound periderm. Peroxidase data imply that there are distinct differences between native and wound periderm, though our data do not indicate that changes in peroxidase activity are involved in the development of resistance to periderm abrasion that occurs upon maturation of the periderm. However, we cannot rule out the involvement in this process of peroxidase isozymes that have low affinity for the substrates utilized here.
PMCID: PMC4233851  PMID: 12125762
Guaiacol; histochemistry; pectin; periderm; peroxidase; phellem; phelloderm; phellogen; potato; ruthenium red; Solanum tuberosum L.; wound‐healing
23.  Methanol May Function as a Cross-Kingdom Signal 
PLoS ONE  2012;7(4):e36122.
Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans.
Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain.
We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.
PMCID: PMC3338578  PMID: 22563443
24.  Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata 
Journal of Experimental Botany  2009;60(9):2631-2640.
Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60–120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.
PMCID: PMC2692009  PMID: 19380422
Defence; herbivory; jasmonic acid; Manduca; methanol; Nicotiana; pectin methylesterase; proteinase inhibitor
25.  Differential transcript profiling through cDNA-AFLP showed complexity of rutin biosynthesis and accumulation in seeds of a nutraceutical food crop (Fagopyrum spp.) 
BMC Genomics  2012;13:231.
Buckwheat, consisting of two cultivated species Fagopyrum tataricum and F. esculentum, is the richest source of flavonoid rutin. Vegetative tissues of both the Fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of F. tataricum are ~50 folds of that in seed of F. esculentum. In order to understand the molecular basis of high rutin content in F. tataricum, differential transcript profiling through cDNA-AFLP has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of F. tataricum compared to F. esculentum.
Differential transcript profiling through cDNA-AFLP in seed maturing stages (inflorescence to seed maturation) with 32 primer combinations generated total of 509 transcript fragments (TDFs). 167 TDFs were then eluted, cloned and sequenced from F. tataricum and F. esculentum. Categorization of TDFs on the basis of their presence/absence (qualitative variation) or differences in the amount of expression (quantitative variation) between both the Fagopyrum species showed that majority of variants are quantitative (64%). The TDFs represented genes controlling different biological processes such as basic and secondary metabolism (33%), regulation (18%), signal transduction (14%), transportation (13%), cellular organization (10%), and photosynthesis & energy (4%). Most of the TDFs except belonging to cellular metabolism showed relatively higher transcript abundance in F. tataricum over F. esculentum. Quantitative RT-PCR analysis of nine TDFs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine TDFs (Ubiquitin protein ligase, ABC transporter, sugar transporter) except MYB 118 showed significantly higher expression in early seed formation stage (S7) of F. tataricum compared to F. esculentum. qRT-PCR results were found to be consistent with the cDNA-AFLP results.
The present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. cDNA-AFLP technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of Fagopyrum species. Increased transcript abundance of TDFs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of F. tataricum over F. esculentum but also in nutritional superiority of the former.
PMCID: PMC3441755  PMID: 22686486
Rutin; cDNA-AFLP; Transcript derived fragments; qRT-PCR

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