Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable -glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol, but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1–5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the expiant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.Abbreviations AS acetosyringone - DMRT Duncan's Multiple Range Test - GUS -glucuronidase - T-DNA transferred DNA We wish to thank Dr. Toshihiko Komari, Japan Tobacco Inc. for providing Ayrobacterium tumefaciens strain LBA4404 (pTOK322). Support by the Rockefeller Foundation in the form of a fellowship to R.R.A. and a grant to T.K.H. is acknowledged. This is journal paper number 14,914 from the Purdue University Agricultural Experiment Station.     

Identification of quantitative trait loci associated with rice eating quality traits using a population of recombinant inbred lines derived from a cross between two temperate japonica cultivars

Improved eating quality is a major breeding target in japonica rice due to market demand. In this study, we performed genetic analysis to identify quantitative trait loci (QTLs) that control rice eating quality traits using 192 recombinant inbred lines (RILs) derived from a cross between two japonica cultivars, 'Suweon365' and 'Chucheongbyeo'. We evaluated the stickiness (ST) and overall evaluation (OE) of cooked rice using a sensory test, the glossiness of cooked rice (GCR) using a Toyo-taste meter, and measured the amylose content (AC), protein content (PC), alkali digestion value (ADV), and days to heading (DH) of the RILs in the years 2006 and 2007. Our analysis revealed 21 QTLs on chromosomes 1, 4, 6, 7, 8, and 11. QTLs on chromosomes 6, 7, and 8 were detected for three traits related to eating quality in both years. QTLs for ST and OE were identified by a sensory test in the same region of the QTLs for AC, PC, ADV, GCR and DH on chromosome 8. QTL effects on the GCR were verified using QTL-NILs (near-isogenic lines) of BC(3)F(4-6) in the Suweon365 background, a low eating quality variety, and some BC(1)F(3) lines. Chucheongbyeo alleles at QTLs on chromosomes 7 and 8 increased the GCR in the NILs and backcrossed lines. The QTLs identified by our analysis will be applicable to future marker-assisted selection (MAS) strategies for improving the eating quality of japonica rice.     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.     

Molecular identification of Mango, Mangifera indica L.var. totupura

Mango (>Mangifera indica) belonging to Anacardiaceae family is a fruit that grows in tropical regions. It is considered as the King of fruits. The present work was taken up to identify a tool in identifying the mango species at the molecular level. The chloroplast trnL-F region was amplified from extracted total genomic DNA using the polymerase chain reaction (PCR) and sequenced. Sequence of the dominant DGGE band revealed that Mangifera indica in tested leaves was Mangifera indica (100% similarity to the ITS sequences of Mangifera indica). This sequence was deposited in NCBI with the accession no. GQ927757. ABBREVIATIONS: AFLP - Amplified fragment length polymorphism , cpDNA - Chloroplast DNA, DDGE - Denaturing gradient gel electrophoresis, DNA - Deoxyribo nucleic acid, EDTA - Ethylenediamine tetraacetic acid, HCl - Hydrochloric acid, ISSR - Inter simple sequence repeats, ITS - Internal transcribed spacer, MATAB - Methyl Ammonium Bromide, Na(2)SO(3) - Sodium sulphite, NaCl - Sodium chloride, NCBI - National Centre for Biotechnology Information, PCR - Polymerase chain reaction, PEG - Polyethylene glycol, RAPD - Randomly amplified polymorphic DNA, trnL-F - Transfer RNA genes start codon- termination codon.     

Antioxidative,antimicrobial and cytotoxic effects of the phenolics of Leea indica leaf extract

This study investigated the phytochemical, antioxidative, antimicrobial and cytotoxic effects of Leea indica leaf ethanol extract. Phytochemical values namely total phenolic and flavonoid contents, total antioxidant capacity, DPPH radical scavenging effect, FeCl₃ reducing power, DMSO superoxide scavenging effect and Iron chelating effects were studied by established methods. Antibacterial, antifungal and cytotoxic effects were screened by disk diffusion technique, food poison technique and brine shrimp bioassay, respectively. Results showed the total phenolic content 24.00 ± 0.81 g GAE/100 g, total flavonoid content 194.68 ± 2.43 g quercetin/100 g and total antioxidant capacity 106.61 ± 1.84 g AA/100 g dry extract. Significant (P < 0.05) IC₅₀ values compared to respective standards were recorded in DPPH radical scavenging (139.83 ± 1.40 μg/ml), FeCl₃ reduction (16.48 ± 0.64 μg/ml), DMSO superoxide scavenging (676.08 ± 5.80 μg/ml) and Iron chelating (519.33 ± 16.96 μg/ml) methods. In antibacterial screening, the extract showed significant (P < 0.05) zone of inhibitions compared to positive controls Ampicillin and Tetracycline against Gram positive Bacillus subtilis, Bacillus cereus, Bacillus megaterium, and Staphylococcus aureus and Gram negative Salmonella typhi, Salmonella paratyphi, Pseudomonas aeroginosa, Shigella dysenteriae, Vibrio cholerae, and Escherichia coli. Significant minimum inhibitory concentrations compared to tetracycline were obtained against the above organisms. In antifungal assay, the extract inhibited the growth of Aspergillus flavus, Candida albicans and Fusarium equisetii by 38.09 ± 0.59, 22.58 ± 2.22, and 22.58 ± 2.22%, respectively. The extract showed a significant LC₅₀ value compared to vincristine sulfate in cytotoxic assay. The results evidenced the potential antioxidative, antimicrobial and cytotoxic capacities of Leea inidica leaf extract to be processed for pharmaceutical use.     

水稻QTL分析的研究进展

水稻许多重要的性状是由多基因控制的数量性状,经典的数量遗传学只能把数量性状作为一个整体进行研究。近年来．高密度分子标记连锁图的构建和有效的生物统计学方法的发展使人们对数量性状遗传基础的研究出现了革命性的变化。通过对不同群体内的个体或品系的分子标记基因型和表型数据的共分离分析,能对QTL进行检测和定位。本文对QTL定位的原理和方法进行了介绍,从QTL的数目和效应、上位性效应、QTL基因型与环境的互作、相关性状的QTL以及个体发育不同阶段的QTL等方面对水稻QTL分析的研究进展进行了综述。水稻基因组测序计划已经完成,本文还对基因组时代水稻QTL精细定位和克隆的方法进行了探讨,对QTL分析在水稻育种中的应用前景进行了展望。     

Bioaugmentation of Mesorhizobium cicer,Pseudomonas spp. and Piriformospora indica for Sustainable Chickpea Production

Physiology and Molecular Biology of Plants - Chickpea establishes symbiotic association with Mesorhizobium to fulfill its nitrogen (N) requirement. Integrating chickpea rhizosphere with potential...     

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.     

Comparative proteomic analysis of drought tolerance in the two contrasting Tibetan wild genotypes and cultivated genotype

Background

Drought is one of major abiotic stresses constraining crop productivity worldwide. To adapt to drought stress, plants have evolved sophisticated defence mechanisms. Wild barley germplasm is a treasure trove of useful genes and offers rich sources of genetic variation for crop improvement. In this study, a proteome analysis was performed to identify the genetic resources and to understand the mechanisms of drought tolerance in plants that could result in high levels of tolerance to drought stress.

Results

A greenhouse pot experiment was performed to compare proteomic characteristics of two contrasting Tibetan wild barley genotypes (drought-tolerant XZ5 and drought-sensitive XZ54) and cv. ZAU3, in response to drought stress at soil moisture content 10 % (SMC10) and 4 % (SMC4) and subsequently 2 days (R1) and 5 days (R2) of recovery. More than 1700 protein spots were identified that are involved in each gel, wherein 132, 92, 86, 242 spots in XZ5 and 261, 137, 156, 187 in XZ54 from SMC10, SMC4, R1 and R2 samples were differentially expressed by drought over the control, respectively. Thirty-eight drought-tolerance-associated proteins were identified using mass spectrometry and data bank analysis. These proteins were categorized mainly into photosynthesis, stress response, metabolic process, energy and amino-acid biosynthesis. Among them, 6 protein spots were exclusively expressed or up-regulated under drought stress in XZ5 but not in XZ54, including melanoma-associated antigen p97, type I chlorophyll a/b-binding protein b, glutathione S-transferase 1, ribulosebisphosphate carboxylase large chain. Moreover, type I chlorophyll a/b-binding protein b was specifically expressed in XZ5 (Spots A4, B1 and C3) but not in both of XZ54 and ZAU3. These proteins may play crucial roles in drought-tolerance in XZ5. Coding Sequences (CDS) of rbcL and Trx-M genes from XZ5, XZ54 and ZAU3 were cloned and sequenced. CDS length of rbcL and Trx-M was 1401 bp (the partial-length CDS region) and 528 bp (full-length CDS region), respectively, encoding 467 and 176 amino acids. Comparison of gene sequences among XZ5, XZ54 and ZAU3 revealed 5 and 2 SNPs for rbcL and Trx-M, respectively, with two 2 SNPs of missense mutation in the both genes.

Conclusions

Our findings highlight the significance of specific-proteins associated with drought tolerance, and verified the potential value of Tibetan wild barley in improving drought tolerance of barley as well as other cereal crops.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1657-3) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of conservation and divergence of microsatellites in rice

Studies on microsatellite distribution and divergence in related genomes contribute towards understanding of genome evolution in eukaryotes. Despite the availability of whole genome sequences of four rice genomes, occurrence and significance of microsatellites in the rice genome has remained a relatively unexplored area of research. We have aligned genomes of two rice subspecies i.e. indica and japonica to understand the trends of microsatellite conservation and divergence in the rice genome. Nearly 62% of the indica microsatellites were also found in the japonica genome. Occurrence of microsatellites showed a negative association with that of retrotransposons. Microsatellites repeat unit length and sequence showed direct influence on the microsatellite locus length. Further, microsatellite allele length was also influenced by the sequence characteristics of the neighbouring regions. CCG repeats were most conserved microsatellite sequences across the different syntenic regions in the two rice genomes and often showed association with CpG islands. Our study suggested that microsatellite distribution is not only governed by a balance between replication slippage and point mutations as proposed earlier, but also by the microsatellite motif sequence and characteristics of microsatellite neighbouring regions in the genome. Thus, this study is likely to prove an important reference for understanding the process of microsatellite evolution and dynamics in the two rice subspecies.     

Gene expression profiles in human HepG2 cells treated with extracts of the Tamarindus indica fruit pulp

Tamarindus indicaL. (T. indica) or locally known as asam jawa belongs to the family of Leguminosae. The fruit pulp had been reported to have antioxidant activities and possess hypolipidaemic effects. In this study, we attempted to investigate the gene expression patterns in human hepatoma HepG2 cell line in response to treatment with low concentration of the fruit pulp extracts. Microarray analysis using Affymetrix Human Genome 1.0 S.T arrays was used in the study. Microarray data were validated using semi-quantitative RT-PCR and real-time RT-PCR. Amongst the significantly up-regulated genes were those that code for the metallothioneins (MT1M, MT1F, MT1X) and glutathione S-transferases (GSTA1, GSTA2, GST02) that are involved in stress response. APOA4, APOA5, ABCG5 and MTTP genes were also significantly regulated that could be linked to hypolipidaemic activities of the T. indica fruit pulp.     

Exogenous auxin affects the oxidative burst in barley roots colonized by Piriformospora indica

Beside a cardinal role in coordination of many developmental processes in the plant, the phytohormone auxin has been recognized as a regulator of plant defense. The molecular mechanisms involved are still largely unknown. Using a sensitive chemiluminescence assay, which measures the oxidation of luminol in the presence of H₂O₂ by horseradish peroxidase (HRP), we report here on the ability of exogenously added indole-3-acetic acid (IAA) to enhance the suppressive effect of the root endophyte Piriformospora indica on the chitin-elicited oxidative burst in barley roots. Thus, the potential of P. indica to produce free IAA during the early colonization phase in barley might provide the symbiont with a means to interfere with the microbe-associated molecular patterns (MAMP)-triggered immunity.     

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future.     

Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare)

Background and Aims

Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots.

Methods

Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectic focusing IEF/SDS–PAGE and 2D Blue native BN/SDS–PAGE was studied.

Key Results

The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H₂O₂-producing and consuming peroxidase activities. In contrast, by a factor of 50, higher Mn concentrations did not produce Mn toxicity symptoms on older leaves in rice. Peroxidase activities, lower by a factor of about 100 in the rice leaf AWF compared with barley, support the view of a central role for these peroxidases in the apoplastic expression of Mn toxicity. The high Mn tolerance of old rice leaves could be related to a high Mn binding capacity of the cell walls. Proteomic studies suggest that the lower Mn tolerance of young rice leaves could be related to Mn excess-induced displacement of Mg and Fe from essential metabolic functions.

Conclusions

The results provide evidence that Mn toxicity in barley involves apoplastic lesions mediated by peroxidases. The high Mn tolerance of old leaves of rice involves a high Mn binding capacity of the cell walls, whereas Mn toxicity in less Mn-tolerant young leaves is related to Mn-induced Mg and Fe deficiencies.     

The root endophyte fungus Piriformospora indica leads to early flowering,higher biomass and altered secondary metabolites of the medicinal plant,Coleus forskohlii

This study was undertaken to investigate the influence of plant probiotic fungus Piriformospora indica on the medicinal plant C. forskohlii. Interaction of the C. forskohlii with the root endophyte P. indica under field conditions, results in an overall increase in aerial biomass, chlorophyll contents and phosphorus acquisition. The fungus also promoted inflorescence development, consequently the amount of p-cymene in the inflorescence increased. Growth of the root thickness was reduced in P. indica treated plants as they became fibrous, but developed more lateral roots. Because of the smaller root biomass, the content of forskolin was decreased. The symbiotic interaction of C. forskohlii with P. indica under field conditions promoted biomass production of the aerial parts of the plant including flower development. The plant aerial parts are important source of metabolites for medicinal application. Therefore we suggest that the use of the root endophyte fungus P. indica in sustainable agriculture will enhance the medicinally important chemical production.     

Occasional males in parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae): low Wolbachia titer or incomplete coadaptation?

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. Some populations of the parasitoid wasp Asobara japonica are infected with Wolbachia and reproduce parthenogenetically, while other populations are not infected and reproduce sexually. Wolbachia-infected A. japonica females regularly produce small numbers of male offspring. Because all females in the field are infected and infected females are not capable of sexual reproduction, male production seems to be maladaptive. We investigated why these females nevertheless produce males. We tested three hypotheses: high rearing temperatures could result in higher offspring sex ratios (more males), low Wolbachia titer of the mother could lead to higher offspring sex ratios and/or the Wolbachia infection is of relatively recent origin and not enough time has passed to allow complete coadaptation between Wolbachia and host. In all, 33% of the Wolbachia-infected females produced males and 56% of these males were also infected with Wolbachia. Neither offspring sex ratio nor male infection frequency was significantly affected by rearing temperature or Wolbachia concentration of the mother. The mitochondrial DNA sequence of one of the uninfected populations was identical to that of two of the infected populations. Therefore, the initial Wolbachia infection of A. japonica must have occurred recently. Mitochondrial sequence variation among the infected populations suggests that the spread of Wolbachia through the host populations involved horizontal transmission. We conclude that the occasional male production by Wolbachia-infected females is most likely a maladaptive side effect of incomplete coevolution between symbiont and host in this relatively young infection.     

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.