In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase

Agrobacterium tumefaciens-transformed tobacco suspension-cultured cells (TSCC) exhibit no significant quantitative changes of cell wall invertase protein (CWI) during a culture period of 40 days, whereas CWI activity decreases strongly between 10 and 30 days after cell transfer to fresh medium. Western blot analysis revealed that the apoplasmic invertase inhibitor (INH) is equally expressed throughout the entire culture period. When apoplasmic protein fractions from 4 and 28 days old cell cultures are chromatographed on Concanavalin A(ConA)-Sepharose, the non-glycosylated INH always coelutes with the ConA-bound fraction, suggesting that (i) INH and the glycosylated CWI form a complex in the apoplasmic space, and (ii) INH binding is not sufficient for CWI inhibition. The high specificity of INH binding to CWI was confirmed by native cathodic polyacrylamide gel electrophoresis. Expression analysis of CWI and INH indicates that, at least during certain stages of plant development (seedlings, roots of adult plants), CWI activity may be modulated by INH, the latter operating as a regulatory switch.     

Pectin methylesterase inhibitor

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.     

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.     

Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins

Pectin methylesterase (PME) and invertase are key enzymes in plant carbohydrate metabolism. Inhibitors of both enzymes constitute a sequence family of extracellular proteins. Members of this family are selectively targeted toward either PME or invertase. In a comparative structural approach we have studied how this target specificity is implemented on homologous sequences. By extending crystallographic work on the invertase inhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) from Arabidopsis thaliana, we show an alpha-helical hairpin motif to be an independent and mobile structural entity in PMEI. Removal of this hairpin fully inactivates the inhibitor. A chimera composed of the alpha-hairpin of PMEI and the four-helix bundle of Nt-CIF is still active against PME. By contrast, combining the corresponding segment of Nt-CIF with the four-helix bundle of PMEI renders the protein inactive toward either PME or invertase. Our experiments provide insight in how these homologous inhibitors can make differential use of similar structural modules to achieve distinct functions. Integrating our results with previous findings, we present a model for the PME-PMEI complex with important implications.     

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.     

The invertase inhibitor Nt-CIF from tobacco: a highly thermostable four-helix bundle with an unusual N-terminal extension

Plant invertases are sucrolytic enzymes essential for plant metabolism and development. Enzyme activity is regulated on a posttranslational level via inhibitory proteins, referred to as invertase inhibitors. Ectopic expression of invertase inhibitors in crop plants has high biotechnological potential. However, little biochemical and up to now no detailed structural information is available about this class of plant regulatory proteins. Here, we present the crystal structure of the cell wall-associated invertase inhibitor Nt-CIF from tobacco at a resolution of 1.87A. The structural model reveals an asymmetric four-helix bundle with an uncommon N-terminal extension that appears to be critical for the structural integrity of the protein. Structure analysis of a second crystal form grown in the presence of CdCl(2) reveals two metal binding sites. Nt-CIF is highly thermostable and retains full inhibitory activity after cooling to ambient temperatures. The structure of Nt-CIF provides the first three-dimensional information source for the posttranslational regulation of plant invertases. Based on the recently discovered sequence homology between inhibitors of invertases and pectin methylesterases, our structural model is likely to represent a scaffold also used for the regulation of the latter enzymes, which do not share sequence similarity with invertases. Thus, our structural model sets the 3D-stage for the investigation of posttranslational regulation of invertases as well as pectin methylesterases.     

Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.     

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.     

Pectin methylesterase activity of plant DUF538 protein superfamily

DUF538 (domain of unknown function 538) proteins are known as a group of putative hypothetical proteins in a wide range of plant species. They have been identified from some plants challenged with various environmental stresses. However, a little is known about their functional properties. They have been newly predicted to have binding capacity and esterase-type hydrolytic activity towards bacterial lipopolysaccharides and chlorophyll molecules as carboxylic compounds in plants. In the present study, the binding ability and the methylesterase activity of DUF538 proteins towards pectin molecules were also predicted. Their similarities to pectin methylesterases and their binding ability to pectin molecule were predicted using bioinformatic tools as well as the experimental method. A probable cooperation was speculated between DUF538 and pectin methylesterase protein families in cell wall associated defense responses in plants.     

How do pectin methylesterases and their inhibitors affect the spreading of tobamovirus?

After replication in the cytoplasm, viruses spread from the infected cell into the neighboring cells through plasmodesmata, membranous channels embedded by the cell wall. As obligate parasites, viruses have acquired the ability to utilize host factors that unwillingly cooperate for the viral infection process. For example, the viral movement proteins (MP) interacts with the host pectin methylesterase (PME) and both proteins cooperate to sustain the viral spread. However, how and where PMEs interact with MPs and how the PME/MP complexes favor the viral translocation is not well understood. Recently, we demonstrated that the overexpression of PME inhibitors (PMEIs) in tobacco and Arabidopsis plants limits the movement of Tobacco mosaic virus and Turnip vein clearing virus and reduces plant susceptibility to these viruses. Here we discuss how overexpression of PMEI may reduce tobamovirus spreading.     

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.     

A Comparative Genome Analysis of PME and PMEI Families Reveals the Evolution of Pectin Metabolism in Plant Cell Walls

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.     

Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea

Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.     

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation.     

A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors

Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.     

The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor

Attempts to purify the inhibitor of pectin methylesterase (PMEI) from the soluble extract of ripe apricot (Prunus armeniaca) fruit led to isolation of a protein (Pa-INH) similar to PMEI, but having invertase inhibitory activity against vacuolar invertase from tomato. The molecular charge, the native and SDS-PAGE molecular weights were similar to those of PMEI. Partial amino acid sequence indicated a high level of identity with invertase inhibitors and a significant identity with PMEI. Circular dichroism analysis showed a mainly -helix secondary structure for both the inhibitors and a higher thermostability of Pa-INH. Four Cys residues forming disulfide bridges in PMEI were conserved in Pa-INH. Similarly to PMEI, these residues were linked by disulfide bridges (first to second and third to fourth). The free Cys139 of PMEI is substituted by Ala in Pa-INH. The results reported in this study suggest a common structural arrangement of the two inhibitors.