Pectin methylesterase inhibitor cDNA from kiwi fruit

We have newly isolated one partial pectin methylesterase inhibitor (PMEI) and two full-length cDNA clones from a kiwi fruit cDNA library. The two full-length cDNA clones, Adpmei-1 and Adpmei-2, had an open reading frame of 185 amino acids, including a predicted signal peptide sequence necessary for localization in the cell-wall space. As the deduced amino acid sequence of the cloned fragment was almost same as the sequence of the previously purified PMEI protein (Camardella et al., Eur J Biochem 267:4561–4565), the clones were considered to be cDNAs encoding PMEI protein. Southern blot analysis indicated a low-copy number of the PMEI genes. Transgenic analysis of asparagus calli expressing a kiwi fruit PMEI gene driven by the CaMV 35S promoter demonstrated in vivo inhibition effects of PMEI on the endogenous pectin methylesterase (PME) activity. The relative expression levels of the PMEI genes in kiwi fruit, analyzed by competitive PCR, increased with the progression of fruit maturation. Given that PME activity also showed its highest level at the fully ripened stage of maturation, the increase in PMEI expression may not indicate direct inhibitory effects on the PME activity and fruit maturation process.     

Accumulation of putrescine and related amino acids in potassium deficient Sesamum

Sesamum indicum was grown in complete or potassium deficient nutrient solution and amino acids, amines, nitrogen and potassium were determined weekly in the leaves. The incorporation of l-arginine-[U-¹⁴C] into protein was also followed. The interconversions of the amino acids of the ordithine-urea cycle, and their contribution to the formation of amines, were studied in cell-free extracts and intact leaves using labelled amino acids. As the level of potassium in the leaves decreased, the levels of the amino acids ornithine, citrulline and arginine, and of the amines putrescine, N-carbamylputrescine and agmatine increased. Potassium deficiency also reduced the rate of protein synthesis. Putrescine appears to be formed preferentially from citrulline with N-carbamylputrescine as intermediate.     

Mago nashi Interacts with a Taiwania (Taiwania cryptomerioides) Pectin Methylesterase-like Protein

Mago nashi proteins are highly conserved among eukaryotes. They are involved in oogenesis, embryogenesis and germ-line determination during animal development, and play important roles in pollen tube growth, root development and spermatogenesis during plant development. In this study, we used yeast two-hybrid screening to show that the TcMago protein can interact with a Taiwania (Taiwania cryptomerioides) pectin methylesterase-like protein (TcPME1) which consists of a transmembrane domain, a pectin methylesterase inhibitor (PMEI) domain and a pectin methylesterase (PME) domain. The PME domain of TcPME1 was necessary for binding with the TcMago protein. The PME domain was highly conserved in all the plants assayed and had five well conserved active site residues. The predicted protein tertiary structures revealed that the PMEI domain and PME domain of TcPME1 are similar to kiwi (Actinidia deliciosa) PMEI and carrot (Daucus carota) PME, respectively. TcPME1 was expressed abundantly in the early stage of root elongation and accumulated at root tip. Moreover, TcPME1 expression was inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Thus, TcPME1 might be involved in root elongation, shoot development and auxin transport during Taiwania development.     

Antifungal Activity of Crude Extracts of <i>Tectona grandis</i> L.f. against Wood Decay Fungi

Wood is mainly made up of cellulose, hemicelluloses, lignin polymers and other organic and inorganic substances, making it susceptible to deteriorate by various biological agents. Tectona grandis L.f. (Teak) is a timber species with high resistance to biological deterioration, valued for its durability, beauty, and mechanical resistance. The purpose of this work was to evaluate the antifungal activity of crude extracts from teak on various fungi that cause wood deterioration. For this, Teak heartwood was obtained, then fragmented and pulverized until obtaining a flour which was used for compounds extraction using the Soxhlet technique coupled to a rotary evaporator through solvents of increasing polarity (hexane, dichloromethane, tetrahydrofuran, and acetone). The extracts obtained were tested against fungal organisms collected in the field, and the LC₅₀ was determined using teak crude extracts on Artemia salina as a biological model. The results obtained showed that a high flour yield was obtained with hexane (0.951 g), followed by tetrahydrofuran (0.446 g), dichloromethane (0.348 g), and acetone (0.152 g). By using nine fungal organisms that predominantly correspond to the genus Aspergillus, the extractable compounds were tested, inhibiting 25% of mycelial growth with tetrahydrofuran (T. versicolor), and 40.9% with dichloromethane (G. trabeum). Likewise, the biological model of A. salina showed an LC₅₀ of 84.9 μg/mL with hexane, 43.3 μg/mL with dichloromethane, 59.6 μg/mL with tetrahydrofuran, and 54.7 μg/mL with acetone. For this reason, it is concluded that Teak wood contains many extractable compounds in relation to its weight, besides having antimicrobial activity when extracted through polar compounds such as dichloromethane and tetrahydrofuran.     

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.     

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Production of interspecific hybrids between Sesamum alatum Thonn and Sesamum indicum L. through ovule culture and screening for phyllody disease resistance

Sesame (Sesamum indicum L.) is an important oilseed crop grown in the tropical and subtropical regions of the world. Despite the nutritional value and cultural importance, the biotechnological research on sesame is very limited. In this study, we have optimized a simple and efficient protocol for producing an interspecific hybrid between Sesamum alatum and S. indicum through ovule culture. In the cross S. alatum × S. indicum, capsule retention without embryo abortion was extended up to 7 days after pollination by spraying the growth regulator mixture containing 289 µM gibberellic acid (GA₃), 80.6 µM α-naphthalene acetic acid (NAA) and 23.3 µM kinetin. Direct organogenesis was successfully achieved when the ovules, excised from 7-day-old capsules, were cultured on MS medium containing 8.8 µM benzylaminopurine (BAP), 2.8 µM indole acetic acid (IAA) and 1712.3 µM glutamine. The regenerants produced roots on half strength MS medium supplemented with 0.27 µM NAA. Phenotypically, the S. alatum × S. indicum hybrid plants were intermediate to those of parents for majority of the traits. Cytological studies revealed normal meiosis in the hybrid without any chromosomal abnormalities. Peroxidase and esterase isozymes were demonstrated to be useful in the identification of hybrid plants. Screening against phyllody disease under greenhouse conditions revealed that the hybrids were moderately resistant.     

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.     

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.     

Ovicidal activity of Sesamum indicum (L.) oil against Spodoptera littoralis (Boisd.)

In the research for alternative tools and botanical products to control Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Sesamum indicum (L.) (Lamiales: Pedaliaceae) oil was assayed as an ovicide. The mortality increased with existence of fatty acids. Chemical analysis of S. indicum oil using GLC analysis showed palmitic acid as the major fatty acid (51.27%), while the major hydrocarbon and sterols were found to be heneicosane (58.63%) and β-sitosterol (2.60%), respectively. Generally, the values of LC₅₀s indicated that one-day-old egg masses are more susceptible than three-day-old eggs. Also, the leaf dip technique is more efficient than the spraying one. Results showed several features of chorionic surface deformation treated with sesame and KZ oils than control using scanning electron microscopy. Meanwhile, the tested oils caused significant reduction in both total soluble protein and transaminase enzymes as compared to control. Additionally, the oils elongated the incubation period and larval duration than control.     

Induction of cytomixis affects microsporogenesis in Sesamum indicum L. (Pedaliaceae)

Cytomixis was recorded during microsporogenesis in sesame (Sesamum indicum L.), a member of the family Pedaliaceae. The phenomenon of cytomixis was observed at various stages of meiosis in 0.5% sodium azide (SA) treated populations of Sesamum indicum L. Cytomixis was observed to occur through various methods, i.e. by forming cytoplasmic channels and direct fusion of pollen mother cells (PMCs), the former was more frequent than the latter. The migration of nuclear content involved all the chromatin/chromosomes or part of it from donor to recipient cell/cells. Some completely empty meiocytes were also observed. Stickiness, precocious movement, laggards, unorientation and micronuclei were observed in almost all the sets treated with various doses of SA. Increase in the doses of SA had a positive effect on the percentage of PMCs showing cytomixis and chromosomal abnormalities. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous sized pollen grains were observed.     

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.     

Development and characterization of microsatellite markers in Tectona grandis (Linn. f)

Teak (Tectona grandis Linn. f) is a hardwood species of worldwide reputation. It occurs naturally in India, Thailand, Myanmar and Laos. Fifteen highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. Primers to amplify these loci were tested on 265 individual trees from different teak populations. An average of 14.7 alleles per locus were detected and expected heterozygosity ranged from 0.223 to 0.879. These loci constitute a powerful tool in investigating the mating system, gene flow, parentage analysis and population dynamics for use in sustainable management of teak forests and for in situ conservation purposes.     

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa₂CysXaa₂₀CysXaa₂Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation     

Morphological and seed protein characterization of the cultivated and the wild taxa of Sesamum L. (Pedaliaceae)

Seed protein analysis and morphological characterization were carried out in one cultivated and one wild species of Sesamum, Sesamum indicum L. and S. occidentale Regel and Heer. Data on 13 quantitative and 33 qualitative characters of the cultivated species and seven accessions of the wild taxa were analyzed. The genetic diversity of the taxa was assessed using UPGMA dendrogram and one-way ANOVA (p?<?0.05). Principal component analysis (PCA) was executed to identify the significant characters to delimit the taxa. Seed protein analysis showed diverse bands, ranging from 16 to 88?kDa. A dendrogram based on UPGMA analysis of seed protein suggested intraspecific relationships of the wild taxa as evidenced from the morphological characterization.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.