Late embryogenesis abundant proteins (LEA) are associated with desiccation tolerance among photosynthetic organisms and have been reported in mono- and dicot plants as well as in nematodes, yeast, bacteria and cyanobacteria. Although the functional role of LEA proteins remains speculative, there is evidence supporting their participation in acclimation and/or in the adaptive response to stress. EMV1 is a Group 1 LEA protein isolated from Vigna radiata, which is speculated to impart desiccation tolerance in plants. The homology model of this protein was generated by using the LOOPP software based on available structural homologues in protein databases. The final model obtained by molecular mechanics and dynamics method was refined and assessed by PROCHECK and shown to be reliable. The generated model could prove be helpful in understanding functional characteristics of this important class of desiccation tolerance protein
The interactive effects of different levels of NaCl and two biofertilizers on certain aspects of growth and metabolism of lettuce plants were investigated. The addition of a recommended dose of phosphorein biofertilizer to salinized soil, induced significant increases in all growth and reproductive parameters determined in growing lettuce plants. On the other hand, fertigation of such sodic salty soil with a recommended dose of nitrobein biofertilizer, induced slight decreases in the growth and reproductive parameters. The carbohydrate as well as pigment components and the activity of PS II of the salinized lettuce plants fertigated with phosphorein, were increased throughout the three successive growth stages, above the control levels. On the other hand, nitrobein did slight changes in all the metabolites determined, throughout the entire period of the experiment. The results are discussed in relation to applicability of the biofertilizers to sodic salty soil.
The effect of different plat growth regulators like paclobutrazol (PBZ) gibberellic acid and Pseudomonas fluorescens treatments on the non-enzymatic antioxidant components of Catharanthus roseus was investigated in the present study. The treatments were given to plants by soil drenching on 38, 53, 68 and 83 days after planting (DAP). The plants were taken randomly on 45, 60, 75 and 90 DAP and separated into root, stem and leaves and used for determining antioxidant potentials. The non-enzymatic antioxidant contents like total phenol, ascorbic acid (AA), reduced glutathione (GSH) and ?-tocopherol (?-toc), were extracted and assayed from both control and treated plants. It was found that paclobutrazol (PBZ) gibberellic acid and Pseudomonas fluorescens treatments have a profound effect on the antioxidant metabolism and caused an enhancement in non-enzymatic antioxidant potentials under treatments in Catharanthus roseus. Our results have good significance, as this increase the innate antioxidant potential of this medicinal plant, as this plant being an essential component of traditional as well as modern pharmaceutical systems.
The experiments were conducted in earthen pots lined with polythene sheet to find out the effect of different concentrations of cobalt (0, 50, 100, 150, 200 and 250 mg/kg soil) on various morphological parameters, photosynthetic pigment contents and antioxidant enzyme activities on greengram (Vigna radiata (L.) Wilczek). Plants were watered to field capacity daily. Plants were thinned to a maximum of five per pot. The data for various morphological parameters such as, root and shoot length, number of nodules, dry weight of root and shoot and photosynthetic pigments such as chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll and carotenoids content were collected on 30 days after sowing (DAS). Antioxidant enzymes like catalase, peroxidase and polyphenol oxidase activities were analysed from both control and treated plants. All the growth parameters and pigment contents increased at 50 mg/kg cobalt level in the soil, when compared with control. Further increase in cobalt level (100-250 mg/kg) in the soil had a negative impact upon all studied parameters. From these results it is clear that Antioxidant potentials acts as a protective mechanism in Vigna radiata under soil cobalt stress.
Mulberry (Morus) is a deciduous woody tree with moderate tolerance to salinity, which has great economic importance as its leaf is used for feeding the monophagous silkworm Bombyx mori L. Mulberry leaf is also used as fodder for livestock. The fruits of mulberry are highly nutritious and have many medicinal properties. In this review, effects of salinity on the morphological, physiological, biochemical and genetic characters in mulberry were discussed along with recent developments in salt stress research. Since mulberry is moderately resistant to salinity and a great degree of genotypic variation is found, it is possible to develop varieties with enhanced salt tolerance if appropriate strategies and techniques are adopted. An outline of such strategy was also drawn and discussed. Development of salt tolerant varieties can expand mulberry cultivation into the salt affected marginal lands, thereby increasing the availability of leaf for both sericulture and livestock industries in arid and semiarid regions of India and other Asian countries.
In this study the mass of tow Iranian apricot varieties were predicted with using different physical characteristics in four models includes: Linear, Quadratic, S-curve, and Exponential. According to the results, the best and the worst models for prediction the mass of Ghavami cultivar were based on volume and length of the fruit with determination coefficients of 0.80 and 0.61, respectively. Also these results for Rajabali cultivar were based on criteria projected area and length of the fruit with determination coefficients of 0.97 and 0.63, respectively. Also observed that Exponential model was not suitable at all.
The genetic variability of five individuals of Artemisia capillaris from state of Pahang, Malaysia was examined using Inter Simple Sequence Repeat (ISSR) technique. Samples were collected from different regions in Pahang State. Results showed distinctive ISSR banding pattern in this species. Twenty five primers were applied and only ten were selected (807, 809, 825, 834, 841, 862, 866, 876, nIssr 1 and nIssr 3) as reliable amplifying ISSR markers. A total of 90 ISSR bands including 62 polymorphic (68.89%) with amplicon size ranging from 150–2500 bp were scored. Genetic distance for samples ranged from 0.0500 to 0.4200. For similarity index samples were ranged from 0.6400 to 0.9545.
In recent years, questions related to molecular composition and its implications for nutrition and health have been raised as advances in technology speed up the introduction of new diversity into breeding programs, either via transgenic technology or by using molecular markers in combination with wide crosses. Metabolite profiling offers great opportunities for characterization of this diversity phenotypically with respect to its metabolite composition. It provides a powerful resource to guide breeding programs and to alert researchers to positive or detrimental traits at an early stage. The power of this approach will be vastly increased by combining it with transcript profiling and a systematic survey of the metabolite composition of the plant products that are already on the market. This integrated approach and holistic profiling within a systems biology approach enables the careful tracking of the response of the organism to conditional perturbations at different molecular and genetic levels using available databases. This approach to profiling will not only provide a baseline for comparison of plants with novel traits (PNTs) with traditional comparators that are ‘generally recognized as safe’, but also provide a rational framework for risk assessment via 'substantial equivalence'. It also provides important inputs into nutritional research and contributes to the public debate about the acceptability of changes in food-production chains and development of science based regulation of plants with novel traits.
Clonally propagated bermudagrass constitute a major source of turf in the Southern United States. Most of parents for the clonally propagated bermudagrasses were initially introduced from other countries at some time in the past. Few of the cultivars were introduced from China by researchers in the Oklahoma State Agricultural Experimental Station. The objective of this DAF project was to determine the degree of genetic relatedness of these newintroduced Chinese cultivars with the existing vegetative cultivars commonly grown in the United States by using DNA fingerprinting technique (DAF). A total of 89 bands were scored using four DAF primers. The cluster analysis was able to distinguish all the varieties studied into 6 distinct groups indicating the existence of wide genetic variations in the cultivars examined. Tifton 10 showed near similarity coefficient to the Chinese cultivars, the cultivar Tifton 10 was directly increased by the plant collection in Shanghai China by Glenn W. Burton. The closer grouping of Tifton 10 with the recently introduced Chinese accessions indicates the existence of similarity of germplasm within China. The cultivar Tifsport a radiation induced mutant from ‘Midiron’ bermudagrass and Tifway,were the most closely related varieties with an SC of 0.94. The two cultivars Tifsport and Tifway which are morphologically undistinguishable showed a very similar banding pattern indicating these cultivars to be nearly identical. The most distinct cultivars in this study were Midlawn and Quickstand which showed a SC of 0.72.
The genetic diversity of two tetraploid wheat species were analyzed by examining gliadin seed storage protein markers at the Gli-A1 and Gli-B1 loci. Samples were collected from Iran, some areas of the Middle East and north of Africa (Egypt) comprising 15 populations of Triticum persicum and Triticum pyramidale. High rate of electrophoretic polymorphism was detected at the loci studied in two selected species. In T. persicum a total of 72 allelic variants at 2 loci were detected, including 41 for Gli-A1 and 31 for Gli-B1 . In the T. pyramidale samples, 50 allelic variants were totally observed. The number of alleles were 28 and 22 for Gli-A1 and Gli-B1 respectively. No null alleles were observed in the T. persicum samples. The total number of allelic variants in Iranian and othercountries samples of T.persicum were distinguished 42 and 30, respectively. In T.pyramidale conversely, the total number of allelic variants for Egyptian samples were 19 where is higher than other selected countries. In T.persicum population No 1 from Noorabad of Iran showed highest level of genetic diversity (A = 3.00, Ne = 2.11, He = 0.63 and GD = 0.51 ). In T.pyramidale population No,7 from cairo of Egypt had highest diversity ( A = 3.00, Ne = 2.50, He = 0.67 and GD = 0.57)
Five cotton cultivars were crossed in a complete diallel to study the inheritance of different polygenic traits. Genotypic differences were found to be significant (P<0.01) for all the characters. Adequacy tests disclosed that data of all the parameters were fully adequate for genetic analysis except bolls per plant, staple length, fibre strength, and fibre fineness, which was partially or not adequate. Additive component of genotypic variation (D) was significant and predominant for plant height, sympodia per plant, staple length and fibre strength, while dominance effects (H1 and H2) were main controlling factors for of monopodia per plant, number of bolls, lint percentage and seed cotton yield. More dominant genes were revealed in the parents for sympodia per plant, lint percentage and seed cotton yield. The values of heritability in narrow sense (h2 n.s) and H2/4H1 demonstrated asymmetrical and unequal distribution of dominant genes in parents for all characters. Plant height, sympodia per plant, staple length and fibre strength exhibited high narrow sense heritability (h2 n.s) due to the presence of additive gene action, whereas, monopodia per plant, number of bolls, lint percentage and seed cotton yield possessed low heritability. The genetic analysis suggested that plant height, sympodia per plant, staple length and fibre strength could be improved through sib family, pedigree and progeny selection, while exploitation of heterosis would be necessary to attain the genetic advancement in monopodia per plant, number of bolls, lint percentage and seed cotton yield.
Successful in vitro plant regeneration, flowering and fruiting were developed in Tomato (Micro-MsK) a model plant for genetic studies using leaf explants. The leaf explants were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations (1.0–3.0 mg/L) of Benzyl amino purine (BAP) and Zeatin (Zt) individually and also in combination with 0.1 mg/L auxins (IAA/NAA). More number of multiple shoots (15.8) formation per explant was found at 2.0 mg/L Zt. Whereas maximum frequency of adventitious shoots were developed from the leaf explants cultured on MS medium augmented with 0.1 mg/L IAA + 2.0 mg/L Zt in comparison to all the concentrations and combinations used. To know the effect of Timentin on regeneration ability, leaf explants were also cultured on Timentin (100 - 400 mg/L) supplemented with IAA (0.1 mg/l) + Zt (2.0 mg/l) combination. High frequency number of multiple shoots followed by in vitro flowering and fruiting were observed. Viable seeds were formed and also showed the normal germination. The protocol developed in the present investigation may significantly contribute to genetic improvement of tomato.
Genetic diversity of 25 accessions of Cymbopogon aromatic grasses including eight species, two hybrids and one mutant strain were analyzed using DNA markers generated by employing 20 primer pairs derived from cDNAs containing simple sequence repeat (SSR) of rice genome. A total of 151 bands were produced ranging from 3 to 12 per primer pair. The polymorphic information content values varied from 0.143 to 0.916 with an average 0.715. Jaccard’s similarity coefficient ranged from 64 to 87% among the paired accessions. The level of diversity among different taxa/accessions observed during the present study was, however, low relative to the diversity level obtained due to RAPD markers in earlier studies. The pattern of genetic diversity neither matched with the known taxonomic classification, nor did it always match with the distribution of chemical constituents of the essential oils available in these accessions. Thus, present investigation though revealed poor correlation between the molecular and chemical diversity, indicating that chemical diversity in medicinal and aromatic species is not only result of genetic variability, but it also depends on a number of other factors. Thus this study may prove useful in several ways in Cymbopogon conservation and breeding programs and in the development of perfect markers though association mapping for genes involved in controlling agronomically important traits.
The research pertaining to the genetic behavior and impact of various quantitative traits on oil contents in sunflower under water stress conditions at reproductive stage in ten accessions (G-5 G-3, G-9, G-33, G-57, G-93, G-128, A-133, A-75 and HBRS-2) of sunflower was conducted following a triplicate randomized complete block design. The genotypes exhibited significant varietal differences among them for all the characters studied. Whorls per head (0.53) and number of leaves (0.66) displayed highest genotypic and phenotypic coefficients of variation respectively. Heritability in broad sense was the maximum for oil contents (0.715±0.346) followed by achene yield per plant (0.499±0.289) with considerable values of genetic advances indicating the involvement of some additive effects in the inheritance of these traits. Total leaf area exhibited the uppermost value of genetic advance (77.088) with moderate heritability (45±2.76). Plant height showed positive and significant genotypic association with leaf area and achene yield. Similarly number of leaves displayed positive and significant correlation (r=0.727*) with total leaf area and oil contents at genotypic level. The parameters stem diameter, head diameter, whorls per head and fertile whorls per head demonstrated positive and significant genotypic and phenotypic relationship between them. The number of whorls per head disclosed positive genetic association (r=0.625*) with oil contents while oil contents were negative associated with hundred achene weight (r= -0.768*). Path analysis based on oil content as dependent variable revealed that number of leaves, total leaf area, stem diameter and achene yield exerted positive direct effects on the oil contents being stem diameter at the top of the list. Stem diameter exposed the highest indirect positive effects on oil contents through head diameter, whorls per head and fertile whorls per head. These studies revealed that the selection of traits positively associated with oil contents and having positive direct and indirect effects on it could be an efficient selection criteria for oil contents in sunflower.
Wheat seed proteins were studied to identify the cultivar–specific proteins using two Korean pre-harvest sprouting wheat cultivars; Jinpum (susceptible) and Keumgang (resistant). Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3.5-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses. These proteins profiles are divided into 9 categories: Metabolism, Storage, Photosynthesis, Amino Acid, Allergy, Stress, Protein Synthesis, Enzyme and, Hypothetical protein. The gluten includes two different components, high molecular weight glutenin subunits and low molecular weight glutenin subunits and gliadins. Some selected protein spots were detected to be (i) gluten, which is responsible for roughness and viscoelasticity for bread making quality (ii) stress proteins (biotic and abiotic) associated with salt, cold, heat tolerance, disease (iii) pathogen related proteins, and (iv) allergenic proteins responsible for allergy in humans, (v) puroindoline- a & b (encoding PinA and PinB gene)that is responsible for grain texture related to baking performance and roughness and other molecular functions such as antibiotic / toxin / antimicrobial activities, that contribute to the defense mechanism of the plant against predators. Moreover, to gain a better understanding of proteome analysis and identify the pre-harvest sprouting responsible proteins, we carried out a comparative proteomic analysis in pre-harvest sprouting wheat seeds between susceptible and resistant cultivars.
Lipid metabolism plays an important role in the mechanism of frost or cold-tolerance in plants. Plant membrane lipids have the tendency to change from gel to liquid-crystalline phase in response to low temperature stress. This process is due to the increased level of lipid desaturation. The responsible components of this process are, among others, the fatty acid desaturases. Controlling the activity of these enzymes affects the amount of polyunsaturated fatty acids on the glycerol backbone and eventually controls the plants sensitivity to low temperature stress. These metabolic processes trigger a series of changes at the transcriptional level, causing differential expression in genes. Numerous approaches towards this process from chemical to the advanced mass spectrometry were taken during the past decades and several of them will be discussed in this minireview. Metabolomics and transcriptomics seem to be the keys towards describing these complex mechanisms and providing the necessary understanding of lipid metabolic response to low temperature stress.
Potato (Solanum tuberosum) late blight caused by oomycete Phytophthora infestans (Pi) is highly destructive to potato yield and cost huge losses each year. Regulation of a network of transcription machinery, controlled by transcription factors (TF’s), is required
to overcome the susceptibility. WRKY TF’s are known to regulate transcription machinery upon biotic and abiotic stresses in different
crop plants. We cloned and characterized a WRKY gene, StWRKY1, from potato cDNA synthesized from Pi infested leaves. StWRKY1
protein localized typically in the nucleus. Overexpression (OE) of StWRKY1 positively regulates Pi resistance as well as drought
tolerance in transgenic potato. The elevated resistance in OE lines was co-related with higher accumulation of pathogenesis-related (PR) genes as compared to untransformed control plants. Interestingly, increased susceptibility of co-suppression (CSP) plants was associated with down regulation of PR genes expression. Moreover, transgenic lines overexpressing StWRKY1 showed tolerance in terms of less rate of water loss, during dehydration assay. Importantly, expression of StWRKY1 was upregulated upon treatment with plant hormones, suggesting its involvement in basal signal transduction pathway. Overall, our findings provided evidence that StWRKY1 positively regulate biotic and abiotic stress resistance thereby modulating plant basal defense networks, thus play a significant role for crop improvement.
Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant's major constituent is a glycyrrhizin. The β-amyrin 11-oxidase catalyzes the sequential two-step oxidation of β-amyrin in C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showed 99% similarity to β-amyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.
Cucurbita moschata is an economically important species worldwide, but little is known about its genic and genomic information. Few molecular markers have been developed for C. moschata so far, which is not sufficient for breeding programs. The aim of this study was to examine the feasibility of development EST-SSR markers from Cucurbita pepo to C. moschata and to evaluate their potential for genetic analysis in C. moschata cultivars. Eighty-two C. pepo EST-SSRs were tested against 29 C. moschata cultivars from different geographical origin in China. As a result, a high proportion (84.14%) of markers was transferable to C. moschata. From them, nineteen markers exhibited polymorphism. A total of 53 alleles were identified with an average of 2.789 alleles per locus in all the tested samples. The polymorphic information content (PIC), the observed heterozygosity (HO), the expected heterozygosity (HE), and the Shannon's information index (I) were estimated to average 0.393, 0.125, 0.478, and 0.760, respectively. Population structure, principal coordinates and phylogenetic analysis all revealed that these 29 C. moschata cultivars tended to group into two subpopulations, which was related to fruit shape rather than geographic origin. Moreover, every subpopulation possessed its own population-specific alleles. This is the first report describing the development of transferable EST-SSR markers in C. moschata and their application to genetic analysis, which will offer an approach for future marker development and marker-assisted breeding in C. moschata.
Although 2,4-di-tert-butylphenol (2,4-DTBP) has demonstrated strong phytotoxic effect on various weedy plants in previous findings, research on its pre-emergence herbicidal activity in the soil is still scanty. The aim of this study was to investigate the effects of two soil types on pre-emergence herbicidal activity and persistence of 2,4-DTBP. The bioassay was carried out in a growth chamber where goosegrass [Eleusine indica (L.) Gaertn.] seeds were sown in different rates of 2,4-DTBP in two soil series under sterilized and non-sterilized soil conditions. Bioassays of each treatment were conducted in four replicates and arranged in completely randomized design. 2,4-DTBP exhibited potent pre-emergence activity as a root inhibitor where it completely inhibited (100% inhibition) of the root growth of E. indica in sandy loam soil at an application rate of 6.14 kg ai/ha. 2,4-DTBP was rapidly detoxified in silt loam soil as a result of high microbial activity where it completely lost its phytotoxicity by giving 100% emergence within 10 weeks even it was applied at an application as high as 20.4 kg ai/ha. However, 2,4-DTBP remained highly phytotoxic in sandy loam soil where it reduced the root and shoot growth by 47 and 36%, respectively, throughout 10 weeks duration of the investigation. The presence of microbes in non-sterilized soil further suggest that soil microbes may modify the chemical structure of the 2,4-DTBP, which in turn decreased its toxicity. The high level of pre-emergence herbicidal activity in conjunction with its biodegradation in silt loam soil imply that 2,4-DTBP may have potential for development as a natural-soil applied herbicide
A novel serine protease inhibitor gene was isolated from Hevea brasiliensis leaves, a RRIT251 cultivar and designated RRIT251 H. brasiliensis protease inhibitor (251Hbpi). Reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate 251Hbpi. A full-length cDNA of 251Hbpi encoded a 70 amino acid protein. 251HbPI is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. The amino acid residues at the active site of 251HbPI were predicted as Met46-Glu47. Multiple alignments of the homologous PI-I family revealed one motif WPELVG of 251HbPI conserved across the family. 251Hbpi was cloned into expression vector pFLAG-ATS and expressed in Escherichia coli strain BL21. Molecular weight of the recombinant 251HbPI (r251HbPI) was approximately 11 kDa. Protease inhibition analysis revealed that r251HbPI inhibited the activity of chymotrypsin and subtilisin A but did not trypsin protease. Moreover, purified r251HbPI protein inhibited Trichophyton rubrum with a minimum inhibitory concentration of 0.7 mg/ml and a minimum fungicidal concentration of 1.4 mg/ml. The specific T. rubrum protease targets of r251HbPI were analyzed by co-immunoprecipitation. r251HbPI interacted with approximate 27 and 61 kDa T. rubrum proteins, suggesting a role in the inhibition of T. rubrum growth. These results suggest that 251HbPI could be a candidate for the development of a novel drug to treat T. rubrum infection.
In vitro protocorm-like bodies (PLBs) of Dendrobium sonia-28 were cryopreserved through an encapsulation-vitrification method. One to two and 3-4mm PLBs were precultured in half-strength semi-solid Murashige and Skoog (MS) medium supplemented with various sucrose concentrations (0, 0.25, 0.5, 0.75 and 1.0M) at different periods (0, 3, 6 and 9 days). Precultured PLBs were encapsulated and osmoprotected for 24 hours, and then dehydrated in plant vitrification solution 2 (PVS2, 0°C) at different periods (0, 30, 60, 90, 120, 150, 180 and 210 minutes) prior to storage in liquid nitrogen (LN, -196°C) for at least 24 hours. After rapid thawing (40±2°C) for two minutes, the beads were unloaded with 1.2M sucrose and then cultured on half-strength semi-solid MS medium devoid of growth regulators. The 2,3,5-triphenyltetrazolium chloride (TTC) assay was used to determine the viability of the treated PLBs after two weeks of recovery. Histological analyses of non-cryopreserved and cryopreserved PLBs were conducted to assess the impact of the cryopreservation procedure on the in vitro PLB cultures. Observations indicated that cryopreserved PLBs underwent anatomical changes expressed as changes in the cell structure, cell wall, nucleus and cytoplasm. The optimised encapsulation-vitrification parameters involved in this study were the preculture of 3-4mm PLBs for six days in 0.5M sucrose, followed by dehydration in PVS2 at 0°C for 150 minutes. Thus, this method was deemed promising for cryopreservation of PLBs of Dendrobium sonia-28.
Female gametophyte cellularization is an important process in ovules development of Pinus tabuliformis Carr., which is regulated and characterized by multiple proteins. To separate the differential expressed proteins in female gametophyte cellularization, 2D-DIGE were used with its high efficiency and repeatability. The ovule proteins were extracted in prophase and anaphase of the process with TCA-acetone and rinsed with ice-cold acetone at least five times. The protein concentration of the sample solutions was adjusted to 1.5 g·L-1 - 2.5 g·L-1, and the pH of the solutions was kept at 8.5 before labeling. A total of 800 proteins were observed on the 2D-DIGE gel using the optimized protocol. Ninety-one differential expressed proteins were separated with 2D-DIGE, while only 62 were separated with 2D-E. Eleven proteins were specific to prophase and 14 proteins were specific to anaphase. During the cellularization process, 45 proteins were up-regulated and 21 proteins were down-regulated. This study established an 2D-DIGE protocol for proteomics research on the development of P. tabuliformis ovules, and provided a technical reference for 2D-DIGE in tissues that are rich in secondary metabolites like P. tabuliformis.
Crape myrtles (Lagerstroemia spp.) represent a large group of woody flowing plants. Despite their high ornamental value and popularity, few genomic sequences and marker resources are available for them. Lagerstroemia indica is one of the most widely cultivated crape myrtle species. In this study, we partially sequenced the genome of L. indica using newly updated 454 sequencing technology. Over 1.2 million high-quality reads in a total length of 837.4 Mb were generated. The average read length was 679 bp. Of the reads, 779,744 (63.2%) were assembled into 65,129 contigs covering a physical length of 93.6 Mb and with N50 contig size of 1,648 bp. The contigs were used to recover microsatellites with repeat motifs of 1-6 bp. A total of 33,026 microsatellites were detected. An SSR primer database was established based on the flanking sequences of the detected microsatellites. A PCR survey of subset of these SSR primers revealed that 89.5% amplified successfully, and 66.7% of the loci were polymorphic. The polymorphic information contents of the polymorphic SSRs ranged from 0.08 to 0.79, with an average value of 0.44. This study provided valuable genomic sequences and marker resources for future genetic studies on Lagerstroemia species.
The advent of high-throughput sequencing (HTS) strengthened our capacity for small RNA (sRNA) discovery. Here, we did a transcriptome-wide survey of sRNAs, mainly focusing on the microRNAs, with organ-specific expression patterns in both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). By using sRNA HTS data generated by 454 sequencing technology, four organs in Arabidopsis (i.e. six-day-old seedling, rosette leaf, flower and silique), and four in rice (shoot apex, leaf, root apex and inflorescence) were investigated. Chromosome-wide distribution patterns of the organ-specific sRNAs were obtained. We found that, in rice, the 21-nt (nucleotide) sRNAs occupy a large portion of the sRNA population highly expressed in inflorescences. In contrast, the sRNAs not expressed in rice floral organ are predominantly 24 nt in length. Through literature mining, ath-miR156d, ath-miR400, ath-miR822 and ath-miR824 were suggested to be involved in seedling development in Arabidopsis, and osa-miR169 induced by drought and high salinity was indicated to regulate leaf growth in rice.-throughput sequencing; kb -kilobases; miRNA -microRNA; NF-Y -nuclear factor Y; nt -nucleotide; PPR -pentatricopeptide repeat; pre-miRNA -precursor microRNA; RISC -RNA-induced silencing complex; RNA Pol II -RNA polymerase II; RPM -reads per million; sRNA -small RNA; TIGR -The Institute for Genome Research.
We cloned the full length 4CL ortholog encoding 4-coumarate:coenzymeA ligase from kenaf (Hibiscus cannabiuns, GenBank Accession No. JX548316) using degenerate primers and RACE (rapid amplification of cDNA ends) method. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic acid, leading to the synthesis of flavonoids and lignin. The 1,704-bp full length of 4CL ortholog had a 1,623-bp open reading frame (ORF) encoding a predicted protein of 540 amino acids. The predicted molecular weight and isoelectric point (pI) of the deduced protein was 59.56 kDa and 6.58, respectively. The sequence of the deduced amino acid shared 57-79% identities with other 4CL sequences. 4CL ortholog had two conserved putative AMP (adenosine monophosphate)-binding motifs, the SSGTTGLPKGV and GEICIRG domains. A BlastP analysis showed that kenaf 4CL ortholog showed 79% identity with ri4CL2 of Rubus idaeus (AAF91309), which is a class I 4CL involved in lignin synthesis. 4CL ortholog showed differential expression in all tissues during the developmental stages and was highly expressed in stem and root tissues. However, the lowest expression of 4CL ortholog was observed in leaf and mature flower tissues. 4CL ortholog was responsive to various stress conditions in the stem tissues of 3-week-old kenaf plants. Wounding caused biphasic expression at 6 h and 24 h after treatment. Taken together, the results of this study contribute to the knowledge of the presence of 4CL ortholog and its possible role in lignin biosynthesis, as well as its differential expression during developmental stages.
Eukaryotic translation initiation factor 5A (eIF5A) proteins, a highly conserved protein family found in all eukaryotic organisms, are involved in translation elongation, mRNA turnover and decay, cell proliferation, programmed cell death, and abiotic stress responses. However, the precise cellular functions of eIF5A proteins are still not fully known, and especially little is known about their interaction partners. In the present study, we report that an eIF5A protein from Tamarix androssowii, TaeIF5A1, can form homodimers with itself, but cannot form heterodimers with other eIF5A proteins. In addition, TaeIF5A1 can specifically interact with xyloglucan endotransglucosylase/hydrolase (XTH) and ADP-ribosylation factor GTPase activator (Arf GAP), suggesting that TaeIF5A1 may share similar functions with these proteins. Moreover, expression of the TaeIF5A1 confers tolerance to oxidative (H2O2), salt (MgCl2), heavy metal (CuSO4, ZnCl2, and CdCl2), and alkali (NaHCO3 and Na2CO3) stresses to transgenic yeast cells, indicating that it plays a role in abiotic stress tolerance. This study provides information useful in revealing the function of TaeIF5A1 in depth.
The role of plant eIF5A proteins in multiple biological processes such as protein synthesis regulation, translation elongation, mRNA turnover, programmed cell death and stress tolerance is well-known. In the present study, we cloned and characterized a PsneIF5A2 from poplar (Populus simonii × P. nigra) leaves. With the support of bioinformatics prediction, the results showed the domain structures of PsneIF5A2, which was predicted by multiple alignment analysis. It has a S1_eIF5A motif, which is an ortholog of eIF5A1 in Arabidopsis. The plant expression vector of PsneIF5A2 was transformed into Arabidopsis using Agrobacterium tumefaciens strain EHA105. Ectopic expression of PsneIF5A2 in Arabidopsis significantly conferred CuSO4 and NaCl stress tolerance. Furthermore, PsneIF5A2-transgenic Arabidopsis exhibited enhanced SOD and POD activities, lower MDA content and electrolyte leakage under CuSO4 and NaCl stress, compared with control plants. Our results suggested that the PsneIF5A2 gene (GenBank No. KC521463) is an excellent candidate for genetic engineering to improve salt and heavy metal tolerance in agricultural plants.
Drought is an abiotic stress that strongly influences plant growth, development and productivity. To understand the drought tolerance mechanism at the protein level in wheat, a differential proteomics study was carried out on young spring wheat leaves of different genotypes in PEG-stressed and rewatered, using two-dimensional polyacrylamide gel electrophoresis (2-DE). A 2-DE pattern with high resolution and good reproducibility was obtained after staining with Coomassie brilliant blue G-250. Using PDQuest software, 600 protein spots were clearly identified from the treatment and control groups with isoelectric points ranging from 4.0 to 7.0. Thirty-eight differentially expressed protein spots were MALDI-TOF/TOF-MS fingerprinted using 2-DE gel and 35 spots were identified by search through the NCBInr database using Mascot software. Of 35 proteins, twenty-one proteins changed in abundance after PEG stress, with 15 proteins up-regulated, whereas 6 proteins down-regulated. Twenty four hour after rewatering, there were 5 proteins up-regulated and 9 proteins down-regulated compared to the well-watered control. Twenty-two differentially expressed proteins were detected in Qingchun 38 and 13 proteins in Abbondanza. They were involved in photosynthesis, protein biosynthesis, energy pathway, carbon metabolism, cell defense, oxidation reduction, transportation and signal transduction. Our proteomics results suggested that drought stress significantly affects wheat photosynthesis.
First-strand cDNA encoding a serine protease inhibitor was synthesized from RNA extracted from Hevea brasiliensis leaves, RRIM600 cultivar. A full-length cDNA of RRIM600 H. Brasiliensis protease inhibitor (600Hbpi) (GenBank accession no. KJ471471) was obtained from reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The primers for 600Hbpi were created from alignments of H. Brasiliensis RRIM600 latex protease inhibitor (Hb-PI) (GenBank accession no. EU295479) and H. Brasiliensis protease inhibitor protein 1 (PI1) (GenBank accession no. AY221985). 600HbPI encodes a 70 amino acid protein and is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. Multiple sequence alignment of homologous PI-I family proteins revealed one motif WPEL of 600HbPI conserved across the PI-I family. The coding region for the active site of 600HbPI was predicted as Met⁴⁶-Glu⁴⁷. 600Hbpi was cloned into the pFLAG-ATS vector. Recombinant 600HbPI was expressed as 11 kDa proteins in Escherichia coli strain BL21. Protease inhibition analysis showed that recombinant 600HbPI is more effective at inhibiting subtilisin A than chymotrypsin but did not inhibit trypsin protease. These results indicate that the recombinant 600HbPI encoded a functional protease inhibitor that specifically targets the chymotrypsin and subtilisin classes of serine proteases.
The heat shock protein 70 (HSP70) molecular chaperones constitute a large family of highly conserved proteins, which are key survival components under stress and normal physiological conditions. Pyropia seriata grows on intertidal rocks, where it is exposed to environmental changes including desiccation and temperature variations. We identified five HSP70 cDNAs from P. seriata transcriptome. An amino acid sequence analysis suggested that PsHSP70a and PsHSP70d are cytosolic HSP70s, whereas PsHSP70b and PsHSP70c are in the mitochondria and PsHSP70e may be transported into the endoplasmic reticulum. Most of the PsHSP70 genes were detected under normal growth and heat stress conditions, except PsHSP70c. Among them, PsHSP70a displayed the strongest response to heat stress. PsHSP70a-transformed Chlamydomonas showed much higher survival and growth rates than those of wild-type under high temperature conditions. These results indicate that the Pyropia genome contains at least five HSP70 genes, and that cytosolic PsHSP70a is involved in high temperature stress tolerance.
The 812HS rice line, a mutant of two-line sterile rice, exhibits leaf photo-oxidation during the tillering-jointing stage since its chloroplasts are susceptible to damage induced by strong sunlight. To determine the proteomic response to light intensity, the 812HS rice line was exposed to natural light and shade (about one fourth natural light), and two-dimensional electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry was used to compare the proteomic expressions in the rice leaves. Although over 1000 reproducible proteins were detected, only 9.61 % of them displayed differential expression with 1.5-fold abundance. A search of the National Center for Biotechnology Information database revealed 53 proteins, 34 down-regulated and 19 up-regulated under highlight. Among these identified proteins, six ones were related to disease and defense, implying the defense and protection mechanism was built under strong light stress. The increase in S -adenosylmethionine synthetase involved in the biosynthesis of the phytohormone ethylene might contribute to the phenotypic modulation from green to etiolation in photo-oxidation-sensitive leaves. These results suggest the existence of a complex regulatory mechanism of the proteomes in photo-oxidation-sensitive 812HS rice under light stress.
Salvia miltiorrhiza Bge. f.alba is a variety of S. miltiorrhiza Bunge which is a well-known and very important traditional chinese medicinal herb. In this study, we successfully induced hairy roots from leaves of S. miltiorrhiza Bge.falba by Agrobacterium rhizogenes ACCC10060. As determined by high performance liquid chromatography (HPLC), these hairy root cultures had the ability to produce salvianolic acid B and salvianic acid A, two main salvianolic acids in S. miltiorrhiza Bge.falba. The effects of ABA and polyamines (PAs, including putrescine (Put), spermidine (Spd) and spermine (Spe)) on salvianolic acids production were studied. The results showed that phenylalanine ammonia-lyase (PAL) activity, salvianolic acid B and salvianic acid A contents increased 1.8, 2.0 and 3.3 times after 80 μmol L-1 ABA treatment for 10 d, 12 d and 10 d, respectively. Similarly, Put was the most effective polyamine among the three PAs, and the effects of Put and Spd for salvianolic acids production were better than that of Spe. Moreover, hairy root cultures treated with 50 mg L-1 Put produced the highest levels of hairy root growth (13.23 g L-1 culture), salvianolic acid B (12.13 mg g-1 DW) and salvianic acid A (3.95 mg g-1u DW) among the three PAs. After treated with the mixture of Put and Spd (50 mg L-1each) for 10 d, 12 d and 10 d respectively, PAL activity, salvianolic acid B and salvianic acid A production were about 1.82, 2.05 and 3.45 times as those in the control, respectively. These findings indicate that exogenous ABA and PAs can enhance two salvianolic acids production in hairy root cultures of S miltiorrhiza Bge.falba.
Diverse RNA-binding proteins (RBPs) have been determined to play a crucial role in post-transcriptional regulation of RNA metabolism during plant response to abiotic stresses. In this report we characterize a rice (Oryza sativa L.) protein, named OsCBP20, which is similar to subunits of a nuclear cap-binding protein 20 (CBP20), it contains a canonical RNA recognition motif (RRM). We compared the survivability of Escherichia coli (BL21) cells transformed with a recombinant plasmid with control E. coli under different concentration NaCl and mannitol. In addition, we also investigated the high temperature (50 °C) impact on survival of E. coil. The OsCBP20 gene was isolated from rice (Oryza sativa L.) cDNA, encoding protein of 243 amino acids with a calculated molecular mass of 28.6 KDa and a PI of 5.14. On the basis of multiple sequence alignment and phylogenetic analysis, OsCBP20 is classified in RRM family. Recombinant 0sCBP20 protein can be highly expressed in E. coli. The 0sCBP20 protein can enhance the tolerance of E. coli recombinant to high salinity, heat, and dehydration, which suggested that OsCBP20 protein, may play a protective role under stressed conditions. Our work provides new evidence on CBP20 response to abiotic stresses.
Thaumatin like protein (TLPs) gene family have the ability to respond to both biotic and abiotic stresses. In this research, gene expression and bioinformatics analysis were applied to reveal the function of TLPs more evidently. Real Time PCR technique was used to compare TLP gene expression between susceptible and resistant cultivars of wheat under the Mycosphaerella graminicola inoculation. Our results indicated the early resistant-related response by TLP up-regulation within 3h after inoculation. In abiotic stress we observed induction of TLP gene expression during salt stress in susceptible cultivar. 50mM NaCl induced rapid induction of TLP within 3h, while 100mM NaCl conferred TLP up-regulation later at 72h. In silico chromosome walking analysis revealed that four loci can be the candidates for conferring tolerance to both biotic and abiotic stresses. In this study, a combination of Real Time PCR technique and bioinformatics tools suggested the possible role of TLP homologs in response to both biotic and abiotic stresses.
MicroRNAs (miRNAs) are known to regulate plant growth and development via regulating gene expression at both transcriptional and post-transcriptional levels. Although several miRNAs have been reported to be associated with abiotic stress responses in plant, systematic investigation of stress-related miRNAs and their targets in plants is limited. In this study, we systematically investigated stress-related miRNAs and their targets in Arabidopsis thaliana. We identified 94 putative stress-related miRNA genes, in which 8 miRNAs were new identified with stress-related response function based on targets prediction. Sequence analysis of these miRNA genes showed that most stress-related miRNAs possess TATA boxes in their promoters, and more than half contain at least two promoters. We also demonstrated that most stress-related miRNA genes contain stress-related elements in their promoters. Furthermore, conservation analysis showed that many stress-related miRNAs are species/family-specific and a subset of stress-related miRNAs may be derived from repeat sequences. Finally, we found that the stress-related miRNAs target 374 genes with 1,153 predicted target sites, of which 87.2% are targeted for gene cleavage and 12.8% affect protein translation. In conclusion, our findings provide an insight into both the function and evolution of stress-related miRNAs.
In cells, calmodulin (CaM) is the most remarkable Ca2+ transducer. BcCAMTA gene family members are calmodulin-binding transcription activators, which contain new type of sequence-specific DNA-binding domain (CG-1), an ankyrin repeats and tow IQ calmodulin-binding motifs. In our study, 8 calmodulin-binding transcription activator (CAMTA) genes were identified from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino, NHCC), and named as BcCAMTA1, BcCAMTA2.1, BcCAMTA2.2, BcCAMTA3.1, BcCAMTA3.2, BcCAMTA4, BcCAMTA5 and BcCAMTA6 through BcCAMTA genes cloning and according AtCAMTAs. Compared with the classification between Arabidopsis and Chinese cabbage, BcCAMTA family was divided into six subgroups (respectively named as BcCAMTA1-6). Subcellular localization prediction showed that most of the BcCAMTAs were located in the nucleus, except BcCAMTA2.2 and BcCAMTA6 that were located in the cytosol, indicating the different function among BcCAMTAs. The evolution and phylogenetic analysis of BcCAMTAs together with their orthologs from other species showed that CAMTA transcription factor family members duplicated in evolution of species, as well as BcCAMTAs, which showed closer evolutionary relationship with Arabidopsis and Chinese cabbage. Seedlings were exposed to four abiotic stresses including cold, drought, copper ion and nitrate stress to explore the transcriptional levels of BcCAMTA genes. The result exhibited that BcCAMTAs, except for BcCAMTA2, were up-regulated under cold stress in 4 h, among which showed the positive regulation to resist cold stress of NHCC. The expression of BcCAMTA2.1 and BcCAMTA3.2 were found significantly differential expression in five development stages of NHCC, and expressed highest in flowering stage.
The Cys2/His2 (C2H2) type zinc finger (ZF), and Late-embryogenesis abundant (LEA) proteins are associated with various cellular processes that play an important role in plant development and abiotic stress tolerance. The study was designed to evaluate the role of PLEA1:BcZF1 to enhance abiotic stress tolerance in Brassica juncea. The Group 4 LEA, LEA4-1, and ZF proteins isolated from B. napus and B. carinata respectively; were expressed in B. juncea cv. varuna. Expression of ZF protein in B. juncea under the control of LEA promoter showed increased tolerance against multiple abiotic stresses: salt, oxidative and drought. The increased level in the stability of total cellular membrane was observed in the transgenic lines (ZL1, ZL2 and ZL4) of B. juncea. The phenotypic analysis of transgenic lines also showed increased level of root and shoot length as compared to wild type (WT) plants under abiotic stresses. Our study suggest that cDNA encoding BcZF1 and the promoter LEA1 function as regulatory molecules involved in stabilizing and modulating the optimal plant growth under various abiotic stresses.
We cloned a full-length gene from the kenaf plant putatively encoding hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase enzyme (HcHCT), which is involved in the lignin biosynthesis pathway. We examined the tissue and organ specific expression of an HcHCT ortholog during developmental stages and in response to abiotic stresses. The full-length of the HcHCT ortholog consisted of a 1,296 bp open reading frame (ORF) encoding 431 peptides. The molecular weight of deduced amino acids was 47.71 kDa, with an isoelectric point (pI) of 5.79. The deduced amino acid sequence showed 80-86% identities with HCTs of other plants. The deduced amino acid sequence of the HcHCT ortholog has a histidine containing motif (HHAAD), characteristic for acyl transfer catalysis. A second consensus sequence, a DFGWG block, is another acyl transferase of the BAHD family. Phylogenetic analysis showed the closest relationship (86%) with HCT of Populus trichodcarpa (ACC63882). According to quantitative real-time reverse transcription PCR (QPCR) analysis, HcHCT transcript was expressed in all the tissues and organs, but the highest expression was observed in roots and mature flowers. The expression of HcHCT transcript was also examined in stem tissues of 3-week-old kenaf plants in response to various abiotic stresses. The expression of HcHCT transcript was highly induced by all treatments, including wound, SA, NaCl, cold, H2O2, ABA, and drought. HcHCT was highly expressed in response to cold, SA, and H2O2 at 24 h, 6 h, and 6 h after treatment, respectively. Our results suggest that we have cloned the full-length gene putatively encoding for HCT, which is responsive to various abiotic stresses.
Major crop production does not yet match the population growth rate because multiple abiotic stresses hamper the growth and yield of these crops. Most of the plants can tolerate adverse climatic conditions by performing some adaptive machineries but to certain extent. Till date various biotechnological and molecular breeding research approaches have been directed towards developing resistance to single stress factor. However, development of crop plants resistant to a single stress is not an ideal solution in the current agriculture scenario. Occurrence of multiple stresses at a single point of time makes it difficult to formulate research strategies. Considering the huge loss of crop productivity due to these environmental factors, there is an urgent need to direct our research focus towards developing sustainable multi-stress resistance in plants to counter the adverse effect of climate change on the productivity of crops. RNA helicases are ubiquitous proteins that are found in both prokaryotes and eukaryotes. The largest RNA helicase family comprises the DEAD-box RNA helicases which are involved in many aspects of RNA metabolism and in diverse biological processes in plants including regulation of multiple abiotic stress responses. The DEAD-box RNA helicases can be considered as means to identify pathways involved in multiple abiotic stress tolerance. In this review, we summarize the recent advances in elucidating the functions of the DEAD-box RNA helicases in multiple abiotic stress responses and future challenges. We also briefly discussed about our recent research efforts (published and on-going) in this direction. This review would help to formulate new research endeavours utilizing the DEAD-box helicase genes in development of multiple abiotic stress tolerant plants through genetic engineering and biotechnology. Target specific multiplex and multigene CRISPR/Ca9 genome editing would be ideal approach to edit different abiotic stress responsive DEAD-box RNA helicase genes to develop sustainable multiple abiotic stress tolerance in crop plants.
Adenosine triphosphatases (ATPases) belonging to the AAA protein family (ATPases Associated with various cellular Activities) are involved in a wide range of abiotic stress. OsATPase, is one of the gene among these stress genes. However, the knowledge about their roles in developmental processes and response to various stimuli are still very limited in rice. In order to discover new stress tolerance genes in rice (Oryza sativa L.), expression profiles were obtained for leaf and panicle tissues at seedling, booting and heading stages of indica cultivar Pei'ai 64S plants under cold, drought or heat stresses using the GeneChip Rice Genome Array (Affymetrix) representing 51, 279 transcripts from japonica and indica rice. OsATPase was highly expressed in leaves and panicles and in response to cold stress in seedlings and booting stage. Real-time quantitative PCR analysis showed that the result was almost consensus with GeneChip Rice Genome Array, suggesting that OsATPase is a multiple stress responsive gene in rice. In order to study its function in stress tolerance, we cloned the cDNA of the gene through amplification by RT-PCR. Sequence analysis showed that the cDNA encodes a protein of 528 amino acid residues with M.W. 60kD and pI 7.8. Analysis of the putative promoter region for candidate cis-regulatory elements using Plant CARE software identified some cis-elements related to stress responses. Based on the earlier mentioned analysis and results obtained, we propose that OsATPase is a novel candidate gene involved in stress tolerance in rice.
In order to identify the function of a sweet orange (Citrus sinensis [L.] Osbeck cv. Valencia) Alfinl-like (AL) gene, CisAL7, in vitro functional analyses were performed using a procaryotic heterologous expression system (Escherichia coli). CisAL7 was cloned and expressed in a pET28a(+) system. E. coli cells containing the recombinant plasmid or empty vector as a control were treated by multiple stresses. In LB (Luria-Bertani) solid media, Escherichia coli harboring CisAL7 gene was remarkably more tolerant to stresses,s including high salinity (0.6 M NaCl or 0.6 M KCl), high temperature (50°C), and low temperature (4°C), than control cells. These results indicate that CisAL7 protein may play a positive role in responsive to abiotic stresses above. This provides the first experimental evidence that AL7 enhances abiotic tolerance of E. coli cells.
Introgression of alien DNA from wild relatives into cultivated plant genomes by means of interspecific hybridization is a method which has been widely used in breeding. However, the effects of introgression on the patterns of gene transcription of the host genome have rarely been studied. In the present study, in order to understand the molecular defense response of an introgressed line (IL5211S) with introgression from a wild cucumber, Cucumis hystrix, a thaumatin-like gene, referred to as CsPR5, was successfully isolated from IL5211S. The gene was 1504 bp in length with a putative open reading frame of 726 bp, encoding 241 amino acid residues. Open reading frame sequences of CsPR5 from IL5211S and backcross parents were identified. It was also observed that the expression of the CsPR5 gene was enhanced remarkably by DNA introgression of C. hystrix using real-time quantitative PCR (RT-qPCR). In addition, the expressions of CsPR5 in response to Pseudoperonospora cubensis and four different abiotic stresses stimuli (namely salicylic acid, methyl jasmonate, abscisic acid, and hydrogen peroxide) were further analyzed at different time points. These stimuli triggered a significant induction of CsPR5 within 72 h after treatment. These findings indicate that the expression of the CsPR5 gene was enhanced by alien DNA introgression from C. hystrix. This may play a role in the molecular defense of IL5211S against pathogen invasion, and aid in protecting against environmental stresses.
Catharanthus roseus is known as the only source for the low-abundance anticancer agents namely vinblastine and vincristine. Fine tuning of accumulation of such secondary metabolites is highly governed by the regulatory genes. Among these genes, Catharanthus roseus MYC1 (CrMYC1) is known as one of the key transcription factors regulating the biosynthesis of terpenoid indole alkaloid metabolites in C. roseus. In this study, CrMYC1 coding sequence (AF283506) was isolated and cloned in PBI121 plant binary vector. Then, CRMYC1 was transiently overexpressed in C. roseus leaves using agroinfiltration method. In addition to molecular analysis for confirming CrMYC1 overexpression, the profile of some chief terpenoid indole alkaloids in control and transgenic plants was evaluated by HPLC to elucidate the role of CrMYC1 in an increased in the anticancer components. The results indicated that overexpression of CrMYC1 transcription factor can increase most important terpenoid indole alkaloids including vinblastine, vincristine, and catharanthine in C. roseus. HPLC analysis of catharanthine and vinblastine contents showed about 3 and 2.5 fold increases, respectively, while the increase in vinecristine was not significant compared to that of the control. Therefore, CrMYC1 is introduced as an efficient candidate for manipulating TIA pathway in C. roseus and increasing at least the most valuable terpenoid indole alkaloids in this plant.
ISSR and morphological markers were used to detect genetic diversity in several genotypes of Achillea tenuifolia from different geographical regions of Iran. Fifteen primers revealed 247 polymorphic bands, out of which 214 (86.78%) were polymorphic. The dendrogram was constructed using SM coefficient and UPGMA method. The generated dendrogram revealed three groups. The accessions originated from central regions of the country separated from others in group 3. The principle coordinate analysis (PCoA) confirmed the results of clustering (>90%). For morphological traits, North-western (NW) accessions had the highest values of leaf length, leaf width, leaf area, essential oil yield and the latest flowering time, while the Northern one (AtN76) had the highest flower diameter and number of florets in main inflorescence. Results showed the relatively broad genetic base of in most of the accessions evaluated in this study. The lowest and the highest gene diversity were obtained in North-western (AtNW) group (0.18) and Northern (AtN) accession (0.28) respectively. High genetic variation of A. tenuifolia might be attributed to its reproductive propagation and seed dispersal. So, conservation strategies should be provided to maintain such diversity aiming to improve future breeding programs.
Morocco is characterized by its tremendous diversity at all levels for various species, including several crop species, such as durum wheat (Triticum turgidum L. var. durum). The precise identification and characterization of the accessions conserved in the National Genebank of Morocco is of great value for quantifying the extent of genetic diversity within accessions, detecting duplications of genetic materials in germplasm collection, improving and securing durum wheat cultivars in Morocco and in the world. The objective of the present study was to characterize a subset of 23 Moroccan durum wheat accessions held in the genebank for their variability related to quality properties using protein markers of prolamin loci (gliadin and glutenin). The allelic variation at prolamin proteins were studied using polyacrylamide gel electrophoresis namely, A-PAGE and SDS-PAGE. The prolamin differences in the durum accessions were analyzed at the Glu-A1, Glu-A2, Glu-A3/Glu-B3/Glu-B2, and Gli-B1 loci. For the HMW-GS, all the durum wheat accessions studied possess the null subunit at Glu-A1, except for the accession number 4 which possesses the HMW-GS1. At the Glu-B1 alleles, results showed that the majority of accessions possess subunits 20x + 20y or subunit 20; accessions 2, 4, and 19 possess subunits 6 + 8; accession 18 possesses subunit 7 and accessions 6, 7, and 22 possess subunits 7 + 8. The electrophoretic data indicated that the evaluated germplasm encompasses useful variations at prolamin loci. Further investigations are in progress to study the genetic variations in Moroccan durum wheat collection using molecular markers.
The plants are constantly exposed to environmental changes that need to be addressed with appropriate modifications of gene expression. The genetic approach to the study of the response to biotic and abiotic stresses is of great importance in plants, for the identification of the genes involved and their activation mechanisms. In our work, we have characterized the expression of some genes induced during the acclimation under low temperatures in plants of Olea europaea L., by isolating DNA sequences differentially expressed in a cold tolerant clone of Leccino cultivar by using the suppression subtractive hybridisation (SSH). The sequences obtained were analysed by sequencing. Some sequences of the libraries and from orthologous genes expressed in various abiotic/biotic stress treatments, were labelled and used as probes for slot blot hybridization with total RNAs extracted from cold sensitive and tolerant Leccino clones treated with decreasing temperatures down to -10 °C, thus mimicking a process of cold acclimation. Six genes were found to be of interest for the adaptation to cold stress, whose expression is differentially modulated in the two clones. The most important result was in a bimodal response during the acclimation phases, the causes of which have been discussed.
Saussurea laniceps is a perennial herbal alpine plant possessing strong cold tolerance. To get a deeper insight into its cold tolerance mechanisms, freezing tolerance and the proteomic profiles of cold-acclimated S. laniceps plantlets were analyzed. The survival rate of plantlets with height of 8-9 cm were recorded after exposure to chilling stress (2 °C) and compared to plant material kept at a control temperature (23 °C). The survival percent at-10°C increased from 0% to 40% during the 12 d of acclimation. Proteomic analyses, by two-dimensional gel electrophoresis (2-DE), performed during this stage revealed that 9 proteins were up-regulated, while 5 proteins were down-regulated. Among the proposed accumulating or appearing proteins, chlorophyll a-b binding protein 2 (LHCP-2), ribulose-1, 5-bisphosphate carboxylase/oxygenase activase (RCA), 33kDa manganese stabilizing chloroplast protein (33kDa MSP), and oxygen-evolving complex protein 1(OEC-1) were all related to photosynthesis, Maturase K is involved in gene expression regulation and galactinol synthase (GS) related to raffinose synthesis. Cold exposure induced a decrease in the candidate proteins including microtubule plus-end binding protein, ribosomal protein S13, O-acetylserine (thiol) lyase and photosystem I assembly protein ycf4. These results suggest changes in proteins associated with energy production, microtubule dynamic, raffinose synthesis and gene expression regulation process allow S. laniceps to enhance its freezing tolerance in the chilling environment.
Improving drought tolerance through drought preconditioning is an important way to understand drought tolerance mechanism in plants. Some limited researches have focused on the gene expression patterns in conjunction with the underlying enzymes promoting drought tolerance and post-drought recovery in white clover. The objective of this study was to identify whether preconditioning-induced drought tolerance is in relation to changes of antioxidant enzyme activities, gene expression and osmoregulatory solutes in white clover. Plants of white clover cultivar, 'Chuanyin Ladino' (drought sensitive), were exposed to two cycles of drought preconditioning (non-preconditioned, well-watered plants, as a control) then exposed to drought stress for 11 days and rewatered for 6 days in a growth chamber with 14 h photoperiod at day/night temperature of 21/16 °C, 70 % relative humidity, and 300 μimol m-2 s-1 photosynthetic photon flux density. Results showed that drought preconditioning improved drought tolerance and post-drought recovery in white clover, as demonstrated by significantly lower (p≤0.05) lipid peroxidation, better cell membrane stability and higher relative water content in drought-preconditioned plants as compared to non-preconditioned plants under drought stress and rewatering. Drought-preconditioned plants exhibited higher superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase activities (POD), as well as higher transcript level of Cu/Zn SOD gene. Meanwhile, more accumulation of soluble sugars and betain and less accumulated proline were also observed in preconditioned plants. Enhanced antioxidant enzyme activities and significantly higher transcript level of Cu/Zn SOD gene may be the critical reasons in acquiring drought tolerance through drought preconditioning. This study also suggests that drought preconditioning improved white clover drought tolerance, which could be related to more accumulation of soluble sugars and betain, while the accumulated proline is associated with the degree of drought stress injury in white clover.
In vitro hardening or acclimatisation is one of the most practices in plant tissue culture, especially medicinal plant namely Artemisia annua (qinghao), which is very sensitive to ex vitro environments in transplanting process. The aim of this investigation was to enhance the production of artemisinin, sesquiterpene bioactive compound to cure parasite as a major antimalarial drug, and develop a high photosynthetic rate in qinghao plantlets, using photoautotrophic CO 2-enrichment. The artemisinin content in acclimatised plantlets grown in liquid MS medium with vermiculite supporting material was increased when compared to controlled plantlets. Net photosynthetic rate (P n) and growth characters were greatest in the acclimatized plantlets grown in liquid medium with 65±5% relative humidity, leading to the highest survival percentage (81.7%) when transplanted to ex-vitro conditions. In addition, the Pn and growth performance of hardened plantlets in enriched CO 2 condition were better than those of hardened plantlets without CO 2 enrichment, leading to an improvement in the ex-vitro survival percentage (91.7%). Healthy plantlets of qinghao can be produced effectively in vitro, using a liquid medium with CO 2 enrichment in photoautotrophic conditions, resulting in plants to survive in exvitro environments, as well as having effective artemisinin accumulation.