Differential gene expression analysis reveals the fast-growth mechanisms in Melia dubia at different stand ages.

BACKGROUND: Melia dubia Cav. is a fast-growing multipurpose tree suitable for agroforestry and has been widely cultivated for wood-based industries, particularly pulp and paper production. Despite its high economic value in India, there is a lack of information regarding the molecular mechanism driving its fast-growth. Therefore, this study aimed to elucidate the molecular mechanisms responsible for fast-growth by expression analysis of selective candidate genes. METHODS AND RESULTS: Initially, growth traits were assessed, including tree height and diameter at breast height (DBH), across three different ages (one-year-old, two-year-old, and three-year-old) of M. dubia plantations. Tree volume based on tree height and DBH, was also calculated. The analysis of annual tree height increment revealed that the second-year plantation exhibited the higher increment, followed by first and third years. In contrast, DBH was maximum in third-year plantation, followed by the second and first years. Similarly, annual tree volume increment showed a similar trend with DBH that maximum in the third year, followed by second and first years. Furthermore, a differential gene expression analysis was performed using qRT-PCR on four genes such as Phloem Intercalated with Xylem (PXY), Clavata3/Embryo Surrounding Region-Related 41 (CLE41), 1-aminocyclopropane-1-carboxylic acid synthase (ACS-1) and Hemoglobin1 (Hb1) for downstream analysis. The relative gene expression showed up-regulation of CLE41, ACS-1, and Hb1 genes, while the PXY gene was downregulated across the tree ages. Interestingly, a positive association was observed between tree growth and the expression of the selected candidate genes. CONCLUSION: Our results pave the way for further research on the regulatory mechanisms of genes involved in fast-growth and provide a basis for genetic improvement of Melia dubia.

Whole plant response of Pongamia pinnata to drought stress tolerance revealed by morpho-physiological, biochemical and transcriptome analysis.

BACKGROUND: Pongamia is considered an important biofuel species worldwide. Drought stress in the early growth stages of Pongamia influences negatively on the germination and seedling development. Due to lack of cultivar stability under drought stress conditions, establishment of successful plantation in drought hit areas becomes a major problem. To address this issue, drought stress response of four Pongamia genotypes was studied at morphological, physio-chemical and transcriptome levels. METHODS AND RESULTS: Drought stress was levied by limiting water for 15 days on three months old seedlings of four genotypes. A significant effect of water stress was observed on the traits considered. The genotype NRCP25 exhibited superior morpho-physiological, biochemical drought responses. Also, the genotype had higher root length, photosynthetic pigments, higher antioxidant enzymes and solute accumulation compared to other genotypes. In addition, transcript profiling of selected drought responsive candidate genes such as trehalose phosphate synthase 1 (TPS1), abscisic acid responsive elements-binding protein 2 (ABF2-2), heat shock protein 17 (HSP 17 kDa), tonoplast intrinsic protein 1 (TIP 1-2), zinc finger homeodomain protein 2 (ZFP 2), and xyloglucan endotransglucolase 13 (XET 13) showed only up-regulation in NRCP25. Further, the transcriptome responses are in line with key physio-chemical responses exhibited by NRCP25 for drought tolerance. CONCLUSIONS: As of now, there are no systematic studies on Pongamia drought stress tolerance; therefore this study offers a comprehensive understanding of whole plant drought stress responsiveness of Pongamia. Moreover, the results support important putative trait indices with potential candidate genes for drought tolerance improvement of Pongamia.

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.

Signal transduction through glyco(sphingo)lipids. Introduction and recent studies on glyco(sphingo)lipid-enriched microdomains.

The presence of microdomains enriched in clustered glycosphingolipids (GSLs) at the surface of plasma membranes and liposome membranes, and their functional role in signal transduction, have been suggested by a series of observations, as follows: (1) GSL clusters (patches) are observed by electron microscopy; (2) microvesicles enriched in GSLs and other sphingolipids can be isolated as detergent-insoluble particles by sucrose density gradient ultracentrifugation: (3) such vesicles isolated from B16 melanoma cells contain > 90% of cellular GM3, > 90% of c-Src and Ras, approximately 50% of Rho, and approximately 20 percent of Fak, despite the fact that this vesicle fraction contains only 0.5% of total cellular protein (this fraction is termed "detergent-insoluble GSL-enriched microdomain" (DIGEM)); (4) GM3 in DIGEM can be coimmunoprecipitated with c-Src and Rho, indicating a close association of GM3 with these transducer molecules; (5) stimulation of GM3 in B16 melanoma cells by anti-GM3 antibody or by Gg3 results in change of signal transduction. Thus, GSLs, together with various transducer molecules present at DIGEM, may directly induce signal transduction rather than modulate or modify signal transduction created through receptors of growth factors or hormones as previously observed.

Pectin methylesterase regulates methanol and ethanol accumulation in ripening tomato (Lycopersicon esculentum) fruit.

We provide genetic evidence that the production of methanol in tomato fruit is regulated by pectin methylesterase (PME, EC 3.1.1.11), an enzyme that catalyzes demethoxylation of pectins. The role of PME in methanol production in tomato fruit was examined by relating the tissue methanol content to the PME enzymatic activity in wild-type Rutgers and isogenic PME antisense fruits with lowered PME activity. In the wild-type, fruit development and ripening were accompanied by an increase in the abundance of PME protein and activity and a corresponding ripening-related increase in methanol content. In the PME antisense pericarp, the level of methanol was greatly reduced in unripe fruit, and diminished methanol content persisted throughout the ripening process. The close correlation between PME activity and levels of methanol in fruit tissues from wild-type and a PME antisense mutant indicates that PME is the primary biosynthetic pathway for methanol production in tomato fruit. Interestingly, ethanol levels that were low and unchanged during ripening of wild-type tomatoes increased progressively with the ripening of PME antisense fruit. In vitro studies indicate that methanol is a competitive inhibitor of the tomato alcohol dehydrogenase (ADH, EC 1.1.1.1) activity suggesting that ADH-catalyzed production of ethanol may be arrested by methanol accumulation in the wild-type but not in the PME mutant where methanol levels remain low.

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.

Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora subsp. carotovora that encodes a two-component sensor-regulator protein.

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.

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.

Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits.

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.

Impaired Wound Induction of 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) Synthase and Altered Stem Development in Transgenic Potato Plants Expressing a DAHP Synthase Antisense Construct.

Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification.

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.

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.

Differential expression of tomato (Lycopersicon esculentum L.) genes encoding shikimate pathway isoenzymes. I. 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Tomato (Lycopersicon esculentum L. cv. UC82b) was found to contain two distinct 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase genes that are differentially expressed. The corresponding cDNAs were isolated and characterized. Both genes code for putative plastidic DAHP synthase isoforms. The deduced amino acid sequences are 79% identical. A comparison of the known Solanaceae DAHP synthases indicates two distinct conserved isoforms. The steady-state levels of transcripts of the two tomato genes differ in all organs analysed.

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.

Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.).

Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated.

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits.

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a lambdagtll expression library constructed from MG pericarp poly(A)(+) RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.

Hypopigmented lesions in early leprosy--a clinical and histological study.

26 Patients of leprosy presenting with hypopigmented lesions were divided on morphological grounds into 3 Sub groups, Group I (9 patients) with well-defined single patch with moderate to complete sensory loss; Group II (8 patients) with single ill-defined lesion having partial sensory loss; and Group III (9 patients) having multiple hypo-pigmented patches with mild to moderate sensory loss. Epidermal atrophy was a conspicuous histological finding in all groups. Only patients in Group I showed epitheloid cells in dermal infiltrate with erosion of epidermis in one case. This group may be labelled as maculoanesthetic leprosy. Patients in Group II and III showed mononuclear cell infiltrate in dermis, around neurovascular bundles and appendages. They were histologically consistent with indeterminate leprosy. Follow-up biopsy after six to eight months of treatment showed healing of the lesion of reduction in the infiltrate in most cases.

Wounding induces the first enzyme of the shikimate pathway in Solanaceae.

The first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15), is induced by wounding potato or tomato tissue. The increase in enzyme activity is associated with elevated amounts of the enzyme as determined by immunoblots. The specific wound-induced protein synthesis is preceded by an increase in the mRNA encoding this enzyme. The induced mRNA of potato tuber, leaf, and stem tissue is translated into a precursor polypeptide that is recognized by antibodies raised against the mature enzyme from tuber plastids. Wounding also induces mRNA encoding phenylalanine ammonia-lyase (EC 4.3.1.5), a key enzyme of plant secondary metabolism. The time courses for the induction of the two enzymes are similar, suggesting coordinate regulation for the biosynthesis of primary and secondary aromatic compounds.