[Show abstract][Hide abstract] ABSTRACT: The dietary carotenoids serve as precursor for vitamin A and prevent several chronic-degenerative diseases. The carotenoid profiling is necessary to understand their importance on human health. However, the available high-performance liquid chromatography (HPLC) methods to resolve the major carotenoids require longer analysis times and do not adequately resolve the violaxanthin and neoxanthin.
A fast and sensitive HPLC method was developed using a C30 column at 20°C with a gradient consisting of methanol, methyl-tert-butyl ether and water. A total of 15 major carotenoids, including 14 all-trans forms and one cis form were resolved within 20 min. The method also distinctly resolved violaxanthin and neoxanthin present in green tissues. Additionally this method also resolved geometrical isomers of the carotenoids.
The HPLC coupled with C30 column efficiently resolved fifteen carotenoids and their isomers in shorter runtime of 20 min. Application of this method to diverse matrices such as tomato fruits and leaves, Arabidopsis leaves and green pepper fruits showed the versatility and robustness of the method. The method would be useful for high throughput analysis of large number of samples.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT During plant growth and development, root tip performs multifarious functions integrating diverse external and internal stimuli to regulate root elongation and architecture. It is believed that a signal originating from root tip inhibits lateral root formation (LRF). The excision of root tip induced LRF in tomato seedlings associated with accumulation of auxin in pericycle founder cells. The excision of cotyledons slightly reduced LRF, whereas severing shoot from root completely abolished LRF. Exogenous ethylene application did not alter LRF. The response was modulated by light with higher LRF in seedlings exposed to light. Our results indicate that light plays a role in LRF in seedlings by likely modulating shoot derived auxin.
[Show abstract][Hide abstract] ABSTRACT: Tomato is considered as a model system for Solanaceous plants because of its small genome size and availability of genomic resources. Understanding the pathways controlling different physiological processes and identification of the genes involved in regulating these pathways in tomato (Solarium choperscŕcum) is a challenging task requiring several genetic resources and functional genomic analyses using mutants. Chemical mutagens like ethyl methansulphonate (EMS) which brings about single nucleotide changes, are widely used for developing mutant populations. Here we have generated a comprehensive mutant population of tomato in Indian cultivar Arka Vikas. The EMS mutant population consisting of about 3,80() M2 plants was visually phenotyped by using a Personal Digital Assistant (PDA). Digital recording of all the phenotypic variations were classified intolS major categories and 48 sub categories based on SOL database, “The Genes That Make Tomatoes”. The above mutant population is an excellent resource for both forward as well as reverse genetic studies. Generation of a tomato mutant population with allelic series would aid to functional genomics of plant growth and development.
[Show abstract][Hide abstract] ABSTRACT: In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movements and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning response. Here, we report the isolation and characterization of a Non-phototropic seedling (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark grown seedlings of Nps1mutant phototropin 1 protein accumulates at highly reduced level relative to wild type and lacks BL-induced autophosphorylation. The mutant harbors a single G1484 to A transition in the Hinge1 region of phototropin 1 homologue resulting in an arginine to histidine substitution (R495H) in a highly conserved A'α helix proximal to the LOV2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of A'α helix region in phototropic signaling of tomato.
[Show abstract][Hide abstract] ABSTRACT: Tomato fruit ripening is a complex metabolic process regulated by a genetical hierarchy. A subset of this process is also modulated by light-signaling, as mutants encoding negative regulators of phytochrome signal transduction, show higher accumulation of carotenoids. In tomato phytochromes are encoded by a multi-gene family, namely PhyA, PhyB1, PhyB2, PhyE and PhyF, however, their contribution to fruit development and ripening has not been examined. Using single phytochrome mutants- phyA, phyB1 and phyB2 and multiple mutants- phyAB1, phyB1B2 and phyAB1B2, we compared the on-vine transitory phases of ripening till fruit abscission. The phyAB1B2 mutant showed accelerated transitions during ripening with shortest time to fruit abscission. Comparison of transition intervals in mutants indicated a phase-specific influence of different phytochrome species either singly or in combination on the ripening process. Examination of off-vine ripened fruits indicated that ripening specific carotenoid accumulation was not obligatorily dependent on light and even dark incubated fruits accumulated carotenoids. The accumulation of transcripts and carotenoids in off-vine and on-vine ripened mutant fruits indicated a complex and shifting phase-dependent modulation by phytochromes(s). Our results indicate that in addition to regulating carotenoid levels in tomato fruits, phytochrome(s) also regulate the time required for phase transitions during ripening.
[Show abstract][Hide abstract] ABSTRACT: Tomato (Solanum lycopersicum) high pigment mutants with lesions in diverse loci such as DDB1 (hp1), DET1 (hp2), zeaxanthin epoxidase (hp3) and Ip (Intense pigmentation, gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and the wild type fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast specific carotenoid associated protein (CHRC) in hp1 fruits at red ripe stage which is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper revealed a 2-fold increase in the abundance of CHRC protein in the red ripe stage of hp1 fruits compared with the wild type (WT; Ailsa Craig). CHRC levels in hp2 were found to be similar to that of hp1, whereas, hp3 and Ip showed intermediate level to that in hp1, hp2, and WT fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, zeaxanthin epoxidase and Ip, up-regulates the CHRC levels. Increase in CHRC levels may contribute towards enhanced carotenoid content in these high pigment fruits by assisting in the sequestration and stabilization of carotenoids.
[Show abstract][Hide abstract] ABSTRACT: During many biological experiments voluminous data is acquired, which can be best collected with -portable data acquisition devices and later analyzed with a personal computer (PC). Public domain software catering to data acquisition and analysis is currently limited. The necessity of phenotyping large plant populations led to the development of the application "PHENOME" to manage the data. PHENOME allows acquisition of phenotypic data using a personal digital assistant (PDA) with a built-in barcode scanner. The acquired data can be exported to a customized database on a PC for further analysis and cataloging. PHENOME can be used for a variety of applications, for example high-throughput phenotyping of a mutagenized or mapping population, or phenotyping of several individuals in one or more ecological niches.
[Show abstract][Hide abstract] ABSTRACT: Among the multitude functions performed by plant roots, little
information is available about the mechanisms that allow roots to
overcome the soil resistance, in order to grow in the soil to obtain
water and nutrient. Tomato (Solanum lycopersicum) seedlings grown on
horizontally placed agar plates showed a progressive decline in the root
length with the increasing impedance of agar media. The incubation with
1-methylcyclopropane (1-MCP), an inhibitor of ethylene perception, led
to aerial growth of roots. In contrast, in absence of 1-MCP control
roots grew horizontally anchored to the agar surface. Though
1-MCP-treated and control seedlings showed differential ability to
penetrate in the agar, the inhibition of root elongation was nearly
similar for both treatments. While increased mechanical impedance also
progressively impaired hypocotyl elongation in 1-MCP treated seedlings,
it did not affect the hypocotyl length of control seedlings. The decline
in root elongation was also associated with increased expression of
DR5::GUS activity in the root tip signifying accumulation of auxin at
the root tip. The increased expression of DR5::GUS activity in the root
tip was also observed in 1-MCP treated seedlings, indicating
independence of this response from ethylene signaling. Our results
indicate operation of a sensing mechanism in root that likely operates
independently of ethylene but involves auxin to determine the degree of
impedance of the substratum.
[Show abstract][Hide abstract] ABSTRACT: Plant roots forage the soil for water and nutrients and overcome the soil's physical compactness. Roots are endowed with a mechanism that allows them to penetrate and grow in dense media such as soil. However, the molecular mechanisms underlying this process are still poorly understood. The nature of the media in which roots grow adds to the difficulty to in situ analyze the mechanisms underlying root penetration. Inhibition of ethylene perception by application of 1-methyl cyclopropene (1-MCP) to tomato seedlings nearly abolished the root penetration in Soilrite. The reversal of this process by auxin indicated operation of an auxin-ethylene signaling pathway in the regulation of root penetration. The tomato pct1-2 mutant that exhibits an enhanced polar transport of auxin required higher doses of 1-MCP to inhibit root penetration, indicating a pivotal role of auxin transport in this process. In this update we provide a brief review of our current understanding of molecular processes underlying root penetration in higher plants.
[Show abstract][Hide abstract] ABSTRACT: During seed germination, emerging roots display positive gravitropism and penetrate into the soil for nutrition and anchorage. Tomato (Solanum lycopersicum) seeds germinated in the presence of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, failed to insert roots into Soilrite and grew in the air, forming loops. Time-lapse video imaging showed that 1-MCP-grown root tips retained positive gravitropism and made contact with the surface of Soilrite but failed to penetrate into the Soilrite. Time-course studies revealed that the effect of 1-MCP was most prominent when seed imbibition and germination were carried out in the continual presence of 1-MCP. Conversely, 1-MCP was ineffective when applied postgermination after penetration of roots in the Soilrite. Furthermore, treatment with 1-MCP caused a reduction in DR5::β-glucuronidase auxin-reporter activity and modified the expression of SlIAA3 and SlIAA9 transcripts, indicating interference with auxin signaling. The reduced ethylene perception mutant, Never-ripe, displayed decreased ability for root penetration, and the enhanced polar auxin transport mutant, polycotyledon, showed a nearly normal root penetration in the presence of 1-MCP, which could be reversed by application of auxin transport inhibitors. Our results indicate that during tomato seed germination, a coaction between ethylene and auxin is required for root penetration into the soil.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT
In recent years, mutants have served as useful starting material to unravel the mechanisms governing many biological processes, including both plants and animals. Mainly, there are three ways of inducing mutations; by using physical agents (radiations UV, X-rays,y-rays), chemical agents (Ethyl Methane Sulphonate (EMS), Nitrous Oxide (NO), acridine, etc.) and biological agents (transposon and T-DNA). The mutation induced by these agents lead to isolation of most commonly recessive or uncommonly dominant mutants. The isolation and screening of a mutant are tedious besides being a cumbersome job. Even after isolation, ascertaining the homozygous nature of the plant is equally difficult, especially for a dominant mutant. The commonly known methods for screening of homo/heterozygosity of the mutant are either highly expensive as sequencing of the gene or involve very tedious/time-consuming genetic segregation analysis. Here, we present a rapid tool for identifying the homo/ heterozygous nature of the plants by simple digestion of PCR-amplified genomic DNA with CEL-I endonuclease and resolving the cleaved product on conventional agarose gels. This method is quite robust and provides a great help to researchers in developing countries where instrumentation access is limited.
[Show abstract][Hide abstract] ABSTRACT: Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate.
A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C.
A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.
BMC Research Notes 01/2011; 4:12. DOI:10.1186/1756-0500-4-12
[Show abstract][Hide abstract] ABSTRACT: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection.
The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato.
NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations.
[Show abstract][Hide abstract] ABSTRACT: The data generated during a course of a biological experiment/study can be sometimes be massive and its management becomes quite critical for the success of the investigation undertaken. The accumulation and analysis of such large datasets often becomes tedious for biologists and lab technicians. Most of the current phenotype data acquisition management systems do not cater to the specialized needs of large-scale data analysis. The successful application of genomic tools/strategies to introduce desired traits in plants requires extensive and precise phenotyping of plant populations or gene bank material, thus necessitating an efficient data acquisition system.
Here we describe newly developed software "PHENOME" for high-throughput phenotyping, which allows researchers to accumulate, categorize, and manage large volume of phenotypic data. In this study, a large number of individual tomato plants were phenotyped with the "PHENOME" application using a Personal Digital Assistant (PDA) with built-in barcode scanner in concert with customized database specific for handling large populations.
The phenotyping of large population of plants both in the laboratory and in the field is very efficiently managed using PDA. The data is transferred to a specialized database(s) where it can be further analyzed and catalogued. The "PHENOME" aids collection and analysis of data obtained in large-scale mutagenesis, assessing quantitative trait loci (QTLs), raising mapping population, sampling of several individuals in one or more ecological niches etc.
[Show abstract][Hide abstract] ABSTRACT: Red light, acting via phytochrome, stimulates phenylalanine ammonia lyase (PAL) activity in cotyledons and hypocotyls of tomato seedlings. The time course of photoinduction of PAL activity has a peak level at 4 h after which activity declines significantly. In tomato seedlings PAL activity comprised of three isoforms and light stimulated activity of all three isoforms. A polyclonal antibody raised against PAL purified from tomato leaves recognized PAL protein belonging to PAL-II and PAL-III isoforms. The mode of increase in PAL activity was investigated by immunochemical techniques. The photostimulated increase in PAL activity appeared to be dependent on de novo synthesis of protein and nucleic acid. However, inhibition of protein phosphatase activity blocked increase in PAL activity without affecting the increase in PAL protein levels. The results indicate that in addition to de novo synthesis, the photostimulation of PAL activity likely requires dephosphorylation by a type 2C protein phosphatase.
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that residue 42-57 (TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71 (WFDTGLSEMRLE; recognition sequence 2 or RS-2) in alphaB-crystallin play a role in oligomerization and subunit interaction with alphaA-crystallin. When we created multiple mutations in alphaB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in alphaB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with alphaA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of alphaB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with alphaA-crystallin.
Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between alphaA- and alphaB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified alphaB- and alphaBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label alphaA-crystallin.
The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type alphaB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in alphaB-crystallin. Secondary structural analysis revealed a slight increase in the alpha-helical content of alphaBinvert-crystallin protein as compared to wild-type alphaB-crystallin. Except for an increase in the ellipticity of the alphaBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type alphaB-crystallin. Chaperone-like function was similar in the alphaBinvert-crystallin mutant and wild-type alphaB-crystallin. The inversion of residues decreased the subunit exchange rate with alphaA-crystallin by two fold.
This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in alphaB-crystallin is important for homooligomerization and optimal subunit interaction with alphaA-crystallin.
[Show abstract][Hide abstract] ABSTRACT: Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in αB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with αA-crystallin. To understand the significance of the RS-2 region in interactions between αA- and αB-crystallins, W60R, F61N, and S66G mutants of αB-crystallin were made and tested for their ability to interact with αA-crystallin. W60R and S66G mutations increased the oligomeric size of αB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of αBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of αBF61N was marginally lower than that of αB, whereas the fluorescence intensity of αBW60R decreased by 40% compared with that of αB. The relative availability of hydrophobic sites in the mutants was in the following order: αBS66G ≫ αB = αBF61N = αBW60R. The far-UV CD profiles for the wild type and αB-crystallin mutants indicated no significant changes in their secondary structures, except for αBS66G, which showed an increase in α-helical content. The near-UV CD profiles of αBW60R and αBF61N were nearly similar to that of wild type αB. On the other hand, αBS66G beyond 270 nm exhibited a signature completely different from that of wild type αB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between αB- and αA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between αB- and αA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of αB-crystallin and subunit interaction between αB- and αA-crystallins.
[Show abstract][Hide abstract] ABSTRACT: Amino acid sequences of αB-crystallin, involved in interaction with αA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of αB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified αA-crystallin. Probing the membrane with αA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in αB-crystallin to interact with αA-crystallin. Residues 42-57 and 60-71 interacted more strongly with αA-crystallin than the 88-123 sequence of αB-crystallin. Binding of one of the αB peptides (42-57) to αA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The αB-crystallin sequences involved in interaction with αA-crystallin were distinct from the chaperone sites reported earlier as binding of the αB sequence from residues 42-57 does not alter the chaperone-like function of αA-crystallin. To identify the critical residues involved in interaction with αA-crystallin, R50G and P51A mutants of αB-crystallin were made and tested for their ability to interact with αA-crystallin. The oligomeric size and hydrophobicity of the mutants were, similar. Circular dichroism studies showed that the P51A mutation increased the α-helical content of the protein. While the αBR50G mutant showed chaperone-like activity similar to wild-type αB, αBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with αA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type αB, αB-crystallin peptide (42-57) effectively competed with αBP51A and αBR50G for interaction with αA. Thus, our studies showed that the αB-crystallin sequence (42-57) is one of the interacting regions in αB and αA oligomer formation.