Avtar K. Handa

Chemistry and uses of pectin : A review

Pectin is an important polysaccharide with applications in foods, pharmaceuticals, and a number of other industries. Its importance in the food sector lies in its ability to form gel in the presence of Ca2+ ions or a solute at low pH. Although the exact mechanism of gel formation is not clear, significant progress has been made in this direction. Depending on the pectin, coordinate bonding with Ca2+ ions or hydrogen bonding and hydrophobic interactions are involved in gel formation. In low-methoxyl pectin, gelation results from ionic linkage via calcium bridges between two carboxyl groups belonging to two different chains in close contact with each other. In high-methoxyl pectin, the cross-linking of pectin molecules involves a combination of hydrogen bonds and hydrophobic interactions between the molecules. A number of factors--pH, presence of other solutes, molecular size, degree of methoxylation, number and arrangement of side chains, and charge density on the molecule--influence the gelation of pectin. In the food industry, pectin is used in jams, jellies, frozen foods, and more recently in low-calorie foods as a fat and/or sugar replacer. In the pharmaceutical industry, it is used to reduce blood cholesterol levels and gastrointestinal disorders. Other applications of pectin include use in edible films, paper substitute, foams and plasticizers, etc. In addition to pectolytic degradation, pectins are susceptible to heat degradation during processing, and the degradation is influenced by the nature of the ions and salts present in the system. Although present in the cell walls of most plants apple pomace and orange peel are the two major sources of commercial pectin due to the poor gelling behavior of pectin from other sources. This paper briefly describes the structure, chemistry of gelation, interactions, and industrial applications soft pectin.

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell‐to‐cell movement

Virus‐encoded movement protein (MP) mediates cell‐to‐cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall‐associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME‐binding region resulted in inactivation of TMV cell‐to‐cell movement.

An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit.

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.

Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits.

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.

Nuclear magnetic resonance spectroscopy-based metabolite profiling of transgenic tomato fruit engineered to accumulate spermidine and spermine reveals enhanced anabolic and nitrogen-carbon interactions

Polyamines are ubiquitous aliphatic amines that have been implicated in myriad processes, but their precise biochemical roles are not fully understood. We have carried out metabolite profiling analyses of transgenic tomato (Solanum lycopersicum) fruit engineered to accumulate the higher polyamines spermidine (Spd) and spermine (Spm) to bring an insight into the metabolic processes that Spd/Spm regulate in plants. NMR spectroscopic analysis revealed distinct metabolite trends in the transgenic and wild-type/azygous fruits ripened off the vine. Distinct metabolites (glutamine, asparagine, choline, citrate, fumarate, malate, and an unidentified compound A) accumulated in the red transgenic fruit, while the levels of valine, aspartic acid, sucrose, and glucose were significantly lower as compared to the control (wild-type and azygous) red fruit. The levels of isoleucine, glucose, γ-aminobutyrate, phenylalanine, and fructose remained similar in the nontransgenic and transgenic fruits. Statistical treatment of the metabolite variables distinguished the control fruits from the transgenic fruit and provided credence to the pronounced, differential metabolite profiles seen during ripening of the transgenic fruits. The pathways involved in the nitrogen sensing/signaling and carbon metabolism seem preferentially activated in the high Spd/Spm transgenics. The metabolite profiling analysis suggests that Spd and Spm are perceived as nitrogenous metabolites by the fruit cells, which in turn results in the stimulation of carbon sequestration. This is seen manifested in higher respiratory activity and up-regulation of phosphoenolpyruvate carboxylase and NADP-dependent isocitrate dehydrogenase transcripts in the transgenic fruit compared to controls, indicating high metabolic status of the transgenics even late in fruit ripening.

Characterization and Functional Expression of a Ubiquitously Expressed Tomato Pectin Methylesterase

Pectin methylesterase (PME),a ubiquitous enzyme in plants, de-esterifies the methoxylated pectin in the plant cell wall. We have characterized a PME gene (designated as pmeu1) from tomato (Lycopersicon esculentum) with an expression that is higher in younger root, leaf, and fruit tissues than in older tissues. Hypocotyls and epicotyls show higher accumulation of pmeu1 transcripts compared with cotyledons. pmeu1 represents a single-copy gene in the tomato genome. Comparison of the deduced amino acid sequence of pmeu1 with other PME homologs showed that the N-terminal halves are highly variable, and the C-terminal halves are relatively conserved in plant PMEs. Constitutive expression of a fruit-specific PME antisense gene does not affect the level of pmeu1 transcripts in vegetative tissues but does lower the level of PMEU1 mRNA in developing tomato fruits. These results suggest that there exists developmentally regulated silencing of pmeu1 by a heterologous PME antisense gene. Expression of pmeu1 in tobacco (Nicotiana tabacum) under the control of the cauliflower mosaic virus 35S promoter caused up to a 4-fold increase in PME specific activity that was correlated with the accumulation of PMEU1 mRNA. In vitro transcription-translation analyses show that pmeu1 encodes a 64-kD polypeptide, whereas transgenic tobacco plants expressing pmeu1 accumulate a new 37-kD polypeptide, suggesting extensive posttranslational processing of PMEU1. These results are the first evidence, to our knowledge, of the functional characterization of a PME gene and the extensive modification of the encoded polypeptide.

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene).

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.

Resistance of cultured higher plant cells to polyethylene glycol-induced water stress

Abstract Cell of tomato ( Lycopersicon esculentum Mill, cv. VFNT Cherry) capable of an enhancedability to grow in the presence of water stress were obtained by exposure of cultured cells to a medium containing polyethylene glycol. These cells exhibited a 14-fold greater increase in growth under stress compared to cells never exposed to polyethylene glycol. The increased resistance was stable when the cells were grown in the presence of polyethylene glycol but rapidly was lost when the cells were transferred back to a medium without the stress agent. Cells resistant to polyethylene glycol-induced water stress also showed increased resistance to NaCl. Adaptation is suggested as a likely mechanism by which resistance is increased.

Growth characteristics of NaCl-selected and nonselected cells of Nicotiana tabacum L.

A cell line of Nicotiana tabacum L. var. Wisconsin 38 was selected (S-10 cells) which is capable of growth in medium containing 10 g liter(-1) NaCl. The fresh and dry weights of S-10 cells at stationary phase in medium containing 10 g liter(-1) NaCl were about 60 and 100% respectively, of that of S-10 cells grown without NaCl. When cells normally maintained in the absence of the salt (S-0 cells) were transferred to medium containing 10 g liter(-1) NaCl, they underwent a 14-day lag period before growth could be detected and they reached stationary phase 36 days after inoculation compared to 14 days for S-10 cells. At stationary phase, fresh and dry weights of S-0 and S-10 cells were the same in the presence of salt. The S-0 cells exhibited a reduced growth rate once growth began in medium with 10 g liter(-1) NaCl. The cell mass doubling time of S-0 cells in medium with 10 g liter(-1) NaCl was 4 days compared to 1.2 days for these cells grown in the absence of the salt and 1.6 days for S-10 cells grown in medium with 10 g liter(-1) NaCl. The resistance of the salt-selected cells was stable in the presence of the salt. However, after 5 cell mass doublings following transfer into medium without NaCl, these cells lost their resistance to salt and responded to NaCl like the cell population (S-0 cells) which had not been selected for growth on NaCl.

Maize Storage Proteins: Characterization and Biosynthesis

Developing seeds provide plant molecular biologists with useful model systems for studying the physiological and genetic mechanisms regulating the synthesis of specific plant proteins, i.e. seed storage proteins. These studies have perhaps even greater significance considering the importance of seed proteins in human and livestock nutrition. Maize storage proteins are interesting from both these aspects, since maize is an economically important crop and mutations affecting both the quantitative and qualitative synthesis of maize storage proteins have been identified (Mertz et al., 1964; Nelson et al., 1965). Our research in the last several years has been devoted to the characterization of these storage proteins and the reactions regulating their biosynthesis.