Expression Of Pectin Methylesterase Research Articles

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

The effects of PROTEOLYSIS6 (PRT6) gene suppression on the expression patterns of tomato ethylene response factors

The ubiquitin-mediated proteolysis via Arg N-degron branch regulates various plant developmental processes and responds to multiple stresses. Nevertheless, limited information is available on the significance of the pathway during fruit ripening. This study reports that the suppression of PROTEOLYSIS6 (PRT6) E3 ligase in tomato through RNAi altered the gene expression of four Ethylene Response Factors (ERFs) (SIERF77, SIERF2, JERF1, and JERF3). The transient expression of 35S::PRT6_RNAi and PG::PRT6_RNAi constructs successfully down-regulated the PRT6 expression in leaves and mature green fruits through 35S promoter, and in breaker-stage fruits via polygalacturonase (PG) promoter, respectively. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed an increased expression of SIERF77, SIERF2, and JERF1 but decreased JERF3 expression in the leaves and mature green fruits agroinfiltrated with 35S::PRT6_RNAi. Ripening-specific SIERF2 showed an increased expression in the breaker-stage fruits agroinfiltrated with PG::PRT6_RNAi, supporting the hypothesis that a low expression level of PRT6 affected the expression patterns of ERFs in specific tissues of tomato. Conversely, the expression of pectin methylesterase (PME) decreased in the breaker fruits. The four orthologues of tomato ERFs had been found to be the Arg N-degron substrates in Arabidopsis, with physiological functions being regulated at the protein level. This study showed that the suppression of PRT6 influenced the expression of these four transcription factors and PME.

Boron reduces aluminum deposition in alkali-soluble pectin and cytoplasm to release aluminum toxicity

Boron (B) is indispensable for plant growth and has been reported in the mitigation of aluminum (Al) toxicity in different plants. This study focused on the efficacy of B in reducing Al toxicity to trifoliate orange seedlings in a hydroponic experiment. Boron supply had a positive effect on root length and plant growth-related parameters and attenuated Al-induced inhibition of plasma membrane H+-ATPase activity. X-ray photoelectron spectroscopy (XPS) in conjunction with scanning electron microscope-energy dispersive x-ray spectrometer (SEM-EDS) revealed that B reduced Al accumulation in root cell wall, especially on pectin fractions (alkali-soluble pectin), accompanied by suppressing pectin synthesis, pectin methylesterase (PME) activity and PME expression. Furthermore, B application inhibited NRAT1 expression while increased ALS1 expression, indicating restraining Al transport from external cells to cytoplasm and accelerating accelerating vacuolar sequestration. The results were further demonstrated by transmission electron microscope-energy dispersive x-ray spectrometer (TEM-EDS) analysis. Taken together, our results indicated that B mainly promoted the efflux of H+ by regulating the plasma membrane H+-ATPase activity, and reduced the demethylation of pectin to weaken Al binding to carboxyl. More importantly, B alleviated some of the toxic effects of Al by compartmentalizing Al into vacuoles and decreasing the deposition of Al in cytoplasm.

Transcriptome Analysis Unravels Metabolic and Molecular Pathways Related to Fruit Sac Granulation in a Late-Ripening Navel Orange (Citrus sinensis Osbeck).

Lanelate navel orange (Citrus sinensis Osbeck) is a late-ripening citrus cultivar increasingly planted in China. The physiological disorder juice sac granulation often occurs in the fruit before harvest, but the physiological and molecular mechanisms underlying this disorder remain elusive. In this study, we found that fruit granulation of the late-ripening navel orange in the Three Gorges area is mainly caused by the low winter temperature in high altitude areas. Besides, dynamic changes of water content in the fruit after freezing were clarified. The granulation of fruit juice sacs resulted in increases in cell wall cellulose and decreases in soluble solid content, and the cells gradually became shrivelled and hollow. Meanwhile, the contents of pectin, cellulose, and lignin in juice sac increased with increasing degrees of fruit granulation. The activities of pectin methylesterase (PME) and the antioxidant enzymes peroxidase (POD), superoxide dismutase, and catalase increased, while those of polygalacturonase (PG) and cellulose (CL) decreased. Furthermore, a total of 903 differentially expressed genes were identified in the granulated fruit as compared with non-disordered fruit using RNA-sequencing, most of which were enriched in nine metabolic pathways, and qRT-PCR results suggested that the juice sac granulation is closely related to cell wall metabolism. In addition, the expression of PME involved in pectin decomposition was up-regulated, while that of PG was down-regulated. Phenylalanine ammonia lyase (PAL), cinnamol dehydrogenase (CAD), and POD related to lignin synthesis were up-regulated, while CL involved in cellulose decomposition was down-regulated. The expression patterns of these genes were in line with those observed in low-temperature treatment as revealed by qRT-PCR, further confirming that low winter temperature is associated with the fruit granulation of late-ripening citrus. Accordingly, low temperature would aggravate the granulation by affecting cell wall metabolism of late-ripening citrus fruit.

Salicylic acid reduces the accumulation of aluminum in Panax notoginsen root cell wall pectin via the NO signaling pathway

Salicylic acid (SA) and nitric oxide (NO) are key signal molecules involved in the reduction of Al accumulation in many plants. The purpose of this study was to investigate whether they could reduce the root content of Al and/or its binding to the root cell wall (CW) fractions in Panax notoginseng, and to investigate the corresponding regulatory mechanisms. Effects of Al treatments on the endogenous SA and NO contents of P. notoginseng were detected. Exogenous SA and paclobutrazol (PAC, a SA synthesis inhibitor) treatments were conducted to test the effects on endogenous NO content; exogenous sodium nitroprusside (SNP, a NO doner) and 2-phenyl-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxyde (cPTIO, a NO synthesis inhibitor) treatments were conducted to test the effects on endogenous SA content. Pectin methyltransferase (PMT) and pectin methylesterase (PME) activity and the gene expression, pectin methylesterification degree (PMD), pectin content, and the accumulation of Al in roots were also studied under the treatments described above. Al stress induced a significant increase of the activity and expression of phenylalanine ammonia-lyase (PAL), and promoted the significant increase of endogenous SA content of P. notoginseng roots. The Al promoted accumulation of endogenous NO could be enhanced by exogenous SA treatment, but was reduced by PAC. Accumulation of endogenous NO that promoted by Al could be enhanced by exogenous SA treatment, but was reduced by PAC treatment. However, SNP or cPTIO treatments had no significant effect on the Al-induced endogenous SA content of P. notoginseng. This showed that NO acted downstream of SA signaling of P. notoginseng under Al stress. Al stress can increase the activity and genes expression of PME in the roots of P. notoginseng, reduce PMD, increase CW pectin content, and thus, the binding capacity of CW pectin to Al was enhanced in P. notoginseng roots. Exogenous SA or SNP both reduced the Al-binding capacity of root CW pectin by decreasing the pectin content, and increased the PMD by inhibiting the genes expression and activity of PME. The effects of PAC or cPTIO on the above-mentioned indicators under Al stress were opposite to that of exogenous SA or SNP treatments. The Al stress induced accumulation of pectin and the reduction of PMD in the root CW of P. notoginseng could be reversed by the treatments of exogenous SA or SNP, then the Al contents in root CW pectin were reduced. Al stress activated the endogenous SA and NO signaling pathways in P. notoginseng, and NO acted downstream of SA. It showed that the Al-activated NO-SA signaling pathway contributed in the reduction of Al binding to the root under Al stress.

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol.

We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous enzymes. In this biosensor, methanol and its subsequent metabolism induce expression of a gene encoding a fluorescent protein that was placed under the control of a methanol-inducible promoter. Using quantitative analyses of fluorescence microscopy images, a methanol-inducible promoter and a host strain were selected, and preculture and assay conditions were optimized to improve the methanol detection limit. Fluorescence-activated cell sorting (FACS) analysis of the distribution and geometric mean of cellular fluorescence intensity against various concentrations of methanol revealed a detection limit of 2.5μM. Finally, this biosensor was applied to evaluate the activity of a heterologously expressed pectin methylesterase (PME). The cellular fluorescence intensity was proportional to the copy number of the PME expression cassette, the protein level, and the enzyme activity. This biosensor can be used for high-throughput screening of single cells harboring high methanol-producing activity, and thereby, the development of a bioconversion process using methanol-producing enzymes.

Expression analysis of cell wall assembly and remodelling-related genes in Arabidopsis roots subjected to boron stress and brassinosteroid at different developmental stages

ABSTRACT Plant cell walls are affected by many biotic and abiotic stress conditions. The aim of this study is to determine the effects of 24-Epibrassinolide (EBL) on some cell wall-related genes in root tissue of five- and ten-week-old Arabidopsis thaliana plants exposed to boron (B) deficiency (0 µM) or toxicity (3000 µM) at the transcriptional level. Expressions of the genes that encode cellulose synthase (CESA1, CESA4, CESA6 and CESA8), cellulose synthase-like (CSLB5), expansin (EXPA5, EXPA8 and EXPA14) and cell wall protein (SEB1) decreased under conditions of B deficiency and toxicity. EBL treatments, in general, led the expressions of these genes to reduce significantly. Expressions of xyloglucan endotransglucosylase/hydrolase genes (XTH21 and XTH23) changed only under conditions of B toxicity. Boron stress and/or EBL treatments caused different responses in expression of pectin methylesterase (PME2 and PME41) genes. As a result of B stress, the expression levels of investigated genes changed more in roots of five-week-old plants than in roots of ten-week-old plants. Results of the present study include new findings that support the ability of BRs to increase molecular aspects of tolerance to stress in plants.

Virus-induced gene silencing of pectin methylesterase protects Nicotiana benthamiana from lethal symptoms caused by Tobacco mosaic virus

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30–45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.

High-Resolution Mapping of a Fruit Firmness-Related Quantitative Trait Locus in Tomato Reveals Epistatic Interactions Associated with a Complex Combinatorial Locus

Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Fir(s.p.)QTL2.1 to Fir(s.p.)QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Fir(s.p.)QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Fir(s.p.)QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

The expression of pectin methylesterase in onion flower buds is associated with the dominant male‐fertility restoration allele

With 6 figures and 2 tablesAbstractIn this study, we investigated differential gene expression in the flower buds of male‐fertile and male‐sterile pools by using the cDNA‐sequence‐related amplified polymorphism (SRAP) method and discovered a fragment that was specifically expressed in male‐fertile pool. Cloning and sequence analysis revealed that this differentially expressed sequence corresponded to a pectin methylesterase gene in Allium cepa (AcPME). This sequence was deposited in GenBank (GU384209). In male‐fertility‐restored line, AcPME was specifically expressed in tiny buds but not in roots, leaves and bulbs. In male‐sterile line and maintainer line, AcPME was not expressed in roots, leaves, bulbs or tiny buds. AcPME was differentially expressed during different flowering stages (I, II, III, IV and V). We developed a PCR‐based marker (WHR240) associated with the expression of AcPME. This marker was validated in six other onion lines; in each case, the male‐fertility‐restored onion was reliably identified. WHR240 therefore permits the efficient marker‐assisted selection of maintainer individuals in onion breeding programmes.

Comparison of cell wall metabolism in the pulp of three cultivars of ‘Nanfeng’ tangerine differing in mastication trait

Like sweet orange (Citrus sinensis), tangerine (Citrus reticulata) is another citrus crop grown widely throughout the world. However, whether it shares a common mechanism with sweet orange in forming a given mastication trait is still unclear. In this study, three 'Nanfeng' tangerine cultivars, 'Yangxiao-26' ('YX-26') with inferior mastication trait, elite 'YX-26' with moderate mastication trait and 'Miguang' ('MG') with superior mastication trait, were selected to investigate the formation mechanism of mastication trait. 'MG' had the lowest contents of total pectin, protopectin and lignin and the highest gene expression levels of citrus polygalacturonase (PG) and pectin methylesterase (PME) at the end of fruit ripening, whereas 'YX-26' had the lowest water-soluble pectin (WSP) content, the highest lignin content and the lowest PG and PME expression levels. The contents of cellulose and hemicellulose were similar among the three tangerines. The fruit mastication trait of C. reticulata was determined by the proportions of WSP and protopectin as well as lignin content, not by cellulose and hemicellulose contents. Pectin content could be a major contribution to the feeling of mastication trait, while PG and PME exhibited an important role in forming a given mastication trait according to the present results as well as previous results for C. sinensis.

Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening apple fruit

To elucidate the roles of cell wall-modifying enzymes in apple fruit, we investigated the activity and gene expression of β-galactosidase (β-Gal), α- l-arabinofuranosidase (α- l-Af), polygalacturonase (PG), and pectin methylesterase (PME). Their regulation by ethylene and cold storage (0 °C) was also assessed. ‘Golden Delicious’ and ‘Fuji’ fruit showed differences in their rates of respiration and decline of firmness, as well as demonstrating unique regulated effects. Activities of β-Gal and α- l-Af were higher in ‘Golden Delicious’ than in ‘Fuji’ fruit, although both had similar patterns of change. They were dramatically inhibited by 1-methylcyclopropene (1-MCP) and 0 °C, and enhanced by exposure to ethephon, with stronger response in ‘Golden Delicious’ fruit. Gene expression of cell wall enzymes also was significantly affected by 0 °C, 1-MCP, and ethephon. The difference in α - l-Af expression among treatments in ‘Golden Delicious’ was more significant than in ‘Fuji’ fruit, especially early in storage. In contrast, expression of β-Gal was inhibited by 1-MCP at early stages in ‘Golden Delicious’ fruit and over the entire storage period in ‘Fuji’ fruit, and was significantly enhanced by ethephon treatment in the former but only slightly in the latter. At 0 °C, β-Gal mRNA accumulation was inhibited in both cultivars. PG and PME activities increased during softening, and differed at different stages for each cultivar, and were obviously regulated by ethylene and 0 °C. PME expression was higher in ‘Golden Delicious’ fruit, with far greater differences between cultivars than that detected for PG. Both PME and PG mRNA were more intensively influenced by ethylene and cold storage in ‘Golden Delicious’ fruit. The results confirm that cell wall enzymes play an important role in fruit softening. Of these, β-Gal and α- l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin.

Characterization of changes in pectin methylesterase expression and pectin esterification during tomato fruit ripening

The production, accumulation, and in situ location of pectin methylesterase (EC 3.1.11) was examined in ripening fruit of the processing tomato cv. UC82B. Pectin methylesterase detected with a monoclonal antibody (PME-1) first appeared adjacent to seeds in immature green fruit and was later detected only in tissue adjacent to the cuticle (i.e., exopericarp) during ripening. Enzyme-linked immunosorbant assay and Western blot analysis using PME-1 demonstrated that the fresh-market cultivars Celebrity and Better Boy accumulated lower levels of this immunologically detectable pectin methylesterase during maturation than did processing cv. UC82B, and that the immunologically detected pectin methylesterase and the total detectable pectin methylesterase activities of 'Celebrity' and 'Better Boy' increased throughout ripening. In contrast, processing cv. UC82B displayed a total detectable pectin methylesterase activity profile that peaked during the breaker stage, a finding supported immunologically by tissue-printing. To correlate pectin methylesterase expression during ripening to the degree of methylesterification of pectins in exopericarp cell walls, we subjected exopericarp tissue from 'UC82B' fruit to an immunocytochemical and ultrastructure study. Esterified pectin decreased in some regions of the exopericarp cell walls during fruit development but persisted in some regions as well. Less-esterified pectin was localized in the middle lamella of exopericarp cell walls during preripe stages, while in ripe fruit, this labeling was largely absent.Key words: pectin methylesterase (PME), immunocytochemistry, tissue-print, pectin esterification, Lycopersicon esculentum.

Characterization of changes in pectin methylesterase expression and pectin esterification during tomato fruit ripening

The production, accumulation, and in situ location of pectin methylesterase (EC 3.1.11) was examined in ripening fruit of the processing tomato cv. UC82B. Pectin methylesterase detected with a monoclonal antibody (PME-1) first appeared adjacent to seeds in immature green fruit and was later detected only in tissue adjacent to the cuticle (i.e., exopericarp) during ripening. Enzyme-linked immunosorbant assay and Western blot analysis using PME-1 demonstrated that the fresh-market cultivars Celebrity and Better Boy accumulated lower levels of this immunologically detectable pectin methylesterase during maturation than did processing cv. UC82B, and that the immunologically de- tected pectin methylesterase and the total detectable pectin methylesterase activities of 'Celebrity' and 'Better Boy' in - creased throughout ripening. In contrast, processing cv. UC82B displayed a total detectable pectin methylesterase activity profile that peaked during the breaker stage, a finding supported immunologically by tissue-printing. To corre - late pectin methylesterase expression during ripening to the degree of methylesterification of pectins in exopericarp cell walls, we subjected exopericarp tissue from 'UC82B' fruit to an immunocytochemical and ultrastructure study. Esterified pectin decreased in some regions of the exopericarp cell walls during fruit development but persisted in some regions as well. Less-esterified pectin was localized in the middle lamella of exopericarp cell walls during preripe stages, while in ripe fruit, this labeling was largely absent.

Effects of promoters on the enhancement of pectin methyl esterase expression inAspergillus niger

A pectin methylesterase-encoding gene (pmeA)_has been cloned and transformed intoA. niger wild-type NRRL3. Transformants produced 20-fold more PME than the host strain. For studying the effects of different promoters on thepmeA expression two novel plasmids were constructed, in which thepmeA promoter was replaced by efficient promoters such as theA. nidulans glyceraldehyde-3-phosphate dehydrogenase (pK45) or theA. oryzae α-amylase (pK61) promoter. The highest level of PME expression was achieved with theA. oryzae α-amylase promoter, reaching a 200-fold increase compared to the production by the host strain.