Industrial process proteomics: alfalfa protein patterns during wet fractionation processing

A proteome reference map of major soluble proteins from Medicago sativa (alfalfa) leaves and stems has been established for the first time. Among 195 spots analyzed by mass spectrometry and N-terminal Edman sequencing, 117 spots were unambiguously identified, representing 87 different proteins. Of these 87 proteins, 13 proteins were directly identified from the partial genome of Medicago sativa, 30 from expressed sequenced tags (ESTs) of the model legume Medicago truncatula and 44 from closely relative species by a cross-species protein identification method. The proteome map of Medicago sativa was then set as a reference to study the major high protein content products that are generated during the wet fractionation process of alfalfa green biomass. Using two-dimensional electrophoresis, we studied the variation of the protein patterns at different steps of the industrial-scale process. We clearly show that the process induces significant changes including chemical modifications, proteolytic events, and heat-shock protein responses. Strikingly, a certain level of cellular regulation is conserved during biomass processing, as exemplified by the induction of some heat shock proteins. Finally, all the results obtained in this proteomic study may help to identify novel products and to improve process designs in alfalfa biomass plants.     

转基因培育抗除草剂水稻

以ｐＡＨＣ２０（含Ｂａｒ基因）和ｐＷＲＧ１５１５（含ＧＵＳ基因和潮霉素抗性基因）以及含Ｂａｒ基因和雪莲凝集素（ＧＮＡ）基因的ｐＣＡＭＢＩＡ３３００ ＲＧ为供体ＤＮＡ,选用水稻品系８７２０３、上农香糯及鄂宜１０５的成熟胚诱导出的愈伤组织及微不定芽为受体材料,分别采用基因枪和根癌农杆菌（ＬＢＡ４４０４,含ｐＡＬ４４０４）导入法进行基因转化;经抗性筛选、ＧＵＳ检测和ＰＣＲ分析。结果表明,外源基因已通过基     

QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress

Cell-membrane stability (CMS) is considered to be one of the major selection indices of drought tolerance in cereals. In order to determine which genomic region is responsible for CMS, 104 rice (Oryza sativa L.) doubled haploid (DH) lines derived from a cross between CT9993–5-10–1-M and IR62266-42–6-2 were studied in the greenhouse in a slowly developed drought stress environment. Drought stress was induced on 50-day-old plants by withholding water. The intensity of stress was assessed daily by visual scoring of leaf wilting and by measuring leaf relative water content (RWC). The leaf samples were collected from both control (well-watered) and stressed plants (at 60–65% of RWC), and the standard test for CMS was carried out in the laboratory. There was no significant difference (P>0.05) in RWC between the two parental lines as well as among the 104 lines, indicating that all the plants were sampled at a uniform stress level. However, a significant difference (P<0.05) in CMS was observed between the two parental lines and among the population. No significant correlation was found between CMS and RWC, indicating that the variation in CMS was genotypic in nature. The continuous distribution of CMS and its broad-sense heritability (34%) indicates that CMS should be polygenic in nature. A linkage map of this population comprising of 145 RFLPs, 153 AFLPs and 17 microsatellite markers was used for QTL analysis. Composite interval mapping identified nine putative QTLs for CMS located on chromosomes 1, 3, 7, 8, 9, 11 and 12. The amount of phenotypic variation that was explained by individual QTLs ranged from 13.4% to 42.1%. Four significant (P<0.05) pairs of digenic interactions between the detected QTLs for CMS were observed. The identification of QTLs for this important trait will be useful in breeding for the improvement of drought tolerance in rice. This is the first report of mapping QTLs associated with CMS under a natural water stress condition in any crop plants. Received: 8 September 1999 / Accepted: 13 October 1999     

Rice resistance to blast caused by leaf surface moistening prior to inoculation

Effect of water droplets placed onto rice (Oryza sativa L.) leaves before inoculation with blast fungus Magnaporthe grisea (Hebert) Barr on disease severity and superoxide radical generation by the leaves was investigated. The leaves were inoculated by placement of spore suspension droplets. One day before, droplets of distilled water were placed to the same sites as an inoculum. It was found, that such a pretreatment decreased frequency of susceptible-type lesions by 2 to 2.5 times and increased that of symptomless outcome by 1.5 times in comparison with the nontreated control. Besides, the pretreatment enhanced superoxide radical generation in diffusates of healthy leaves of susceptible cultivar and in diffusates of infected leaves of resistant cultivar one day post inoculation. It is suggested that water contacting with the leaf surface for a rather long time washes out from its cells compounds possessing properties of plant endogenous elicitors. The latter induce superoxide radical formation by plants and, as a consequence, their disease resistance. This may be interpreted as plant adaptation to high humidity, which usually favors infections.     

Retracted: Screening of candidate genes and fine mapping of drought tolerance quantitative trait loci on chromosome 4 in rice (Oryza sativa L.) under drought stress

Due to severe water resource shortage, genetics of and breeding for DT (drought tolerance) in rice (Oryza sativa L.) have become one of the hot research topics. Identification of grain yield QTLs (quantitative trait loci) directly related to the DT trait of rice can provide useful information for breeding new drought‐resistant and water‐saving rice varieties via marker‐assisted selection. A population of 105 advanced BILs (backcross introgression lines) derived from a cross between Zhenshan97B and IRAT109 in Zhenshan97B background were grown under drought stress in a field experiment and phenotypic traits were investigated. The results showed that in the target interval of RM273‐RM255 on chromosome 4, three main‐effect QTLs related to panicle length, panicle number, and spikelet number per panicle were identified (LOD [logarithm of the odds] > 2.0). The panicle length‐related QTL had two loci located in the neighboring intervals of RM17308‐RM17305 and RM17349‐RM17190, which explained 18.80% and 20.42%, respectively, of the phenotypic variation, while the panicle number‐related QTL was identified in the interval of RM1354‐RM17308, explaining 11.47% of the phenotypic variation. As far as the spikelet number per panicle‐related QTL was concerned, it was found to be located in the interval of RM17308‐RM17305, which explained 28.08% of the phenotypic variation. Using the online Plant‐GE query system, a total of 13 matched ESTs (expressed sequence tags) were found in the target region, and of the 13 ESTs, 12 had corresponding predicted genes. For instance, the two ESTs CB096766 and CA765747 were corresponded to the same predicted gene LOC_Os04g46370, while the other four ESTs, CA754286, CB000011, CX056247, and CX056240, were corresponded to the same predicted gene LOC_Os04g46390.     

Genetic diversity and origin of weedy rice (Oryza sativa f. spontanea) populations found in North-eastern China revealed by simple sequence repeat (SSR) markers

BACKGROUND AND AIMS: Weedy rice (Oryza sativa f. spontanea) is one of the most notorious weeds occurring in rice-planting areas worldwide. The objectives of this study are to determine the genetic diversity and differentiation of weedy rice populations from Liaoning Province in North-eastern China and to explore the possible origin of these weedy populations by comparing their genetic relationships with rice varieties (O. sativa) and wild rice (O. rufipogon) from different sources. METHODS: Simple sequence repeat (SSR) markers were used to estimate the genetic diversity of 30 weedy rice populations from Liaoning, each containing about 30 individuals, selected rice varieties and wild O. rufipogon. Genetic differentiation and the relationships of weedy rice populations were analysed using cluster analysis (UPGMA) and principle component analysis (PCA). KEY RESULTS: The overall genetic diversity of weedy rice populations from Liaoning was relatively high (H(e) = 0.313, I = 0.572), with about 35 % of the genetic variation found among regions. The Liaoning weedy rice populations were closely related to rice varieties from Liaoning and japonica varieties from other regions but distantly related to indica rice varieties and wild O. rufipogon. CONCLUSIONS: Weedy rice populations from Liaoning are considerably variable genetically and most probably originated from Liaoning rice varieties by mutation and intervarietal hybrids. Recent changes in farming practices and cultivation methods along with less weed management may have promoted the re-emergence and divergence of weedy rice in North-eastern China.     

Response of rice (Oryza sativa) with root surface iron plaque under aluminium stress

BACKGROUND AND AIMS: Rice (Oryza sativa) is an aquatic plant with a characteristic of forming iron plaque on its root surfaces. It is considered to be the most Al-tolerant species among the cereal crops. The objective of this study was to determine the effects of root surface iron plaque on Al translocation, accumulation and the change of physiological responses under Al stress in rice in the presence of iron plaque. METHODS: The japonica variety rice, Koshihikari, was used in this study and was grown hydroponically in a growth chamber. Iron plaque was induced by exposing the rice roots to 30 mg L(-1) ferrous iron either as Fe(II)-EDTA in nutrient solution (6 d, Method I) or as FeSO(4) in water solution (12 h, Method II). Organic acid in root exudates was retained in the anion-exchange resin and eluted with 2 m HCl, then analysed by high-performance liquid chromatography (HPLC) after proper pre-treatment. Fe and Al in iron plaque were extracted with DCB (dithionite-citrate-bicarbonate) solution. KEY RESULTS AND CONCLUSIONS: Both methods (I and II) could induce the formation of iron plaque on rice root surfaces. The amounts of DCB-extractable Fe and Al on root surfaces were much higher in the presence of iron plaque than in the absence of iron plaque. Al contents in root tips were significantly decreased with iron plaque; translocation of Al from roots to shoots was significantly reduced with iron plaque. Al-induced secretion of citrate was observed and iron plaque could greatly depress this citrate secretion. These results suggested that iron plaque on rice root surfaces can be a sink to sequester Al onto the root surfaces and Fe ions can pre-saturate Al-binding sites in root tips, which protects the rice root tips from suffering Al stress to a certain extent.     

Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells: effects of osmotic cell conditioning and of the physical configuration of plasmid DNA

Microprojectile-mediated transient and integrative transformation frequencies in rice (Oryza sativa cv. Taipei 309) embryogenic suspension cells were studied as a function of various parameters. Mannitol at concentrations of 0.5 and 0.6 m was best for osmotic preconditioning of the cells for transient, but not for integrative transformation, for which sucrose yielded the best and most reliable results. Denaturation of the transforming plasmid DNA prior to bombardment improved transient and integrative transformation frequencies two to three fold. Delivery of double-stranded plasmids in linear form had no effect on transient transformation when compared to supercoiled plasmid DNA, but led to an overall two fold increase in integrative transformation frequency. This shows that optimized protocols for generating transgenic plants should not be based exclusively on transient gene expression assays. Received: 29 September 1997 / Revision received: 27 February 1998 / Accepted: 2 April 1998     

水稻对UV-B辐射增强的抗性遗传及其生理生化特性研究

本研究结果表明紫外线B(UVB,280～330nm)能严重影响水稻秧苗生长发育,从而引起植株矮化,叶片干枯,叶绿素含量降低,单株干物质减少甚至死亡。不同水稻基因型对紫外线的反应存在明显差异。在3个供试品种(HR、M63、Hr)中,HR最抗UVB危害。遗传分析表明水稻抗UVB的性状为数量遗传性状且受多对隐性基因控制,生化分析表明水稻秧苗经UVB处理后,在0～3d内,随着时间推移,秧苗保护酶活性明显增强,之后,保护酶活性逐惭下降,MDA含量相应增加。抗UVB品种HR比感UVB品种Hr保护酶活性一直较高,膜质过氧化程度较低,反映了保护酶对抵抗UVB危害的重要作用。     

Interaction between programmed cell death 5 and calcineurin B-like interacting protein kinase 23 in Oryza sativa

Programmed cell death (PCD) is crucial for plants during development and stress survival. OsPDCD5, an ortholog to mammalian-programmed cell death 5, was previously cloned from rice (Oryza sativa, cv Zhenxian 97A), and its overexpression can induce PCD in transgenic rice. In the present study, immunoblotting analysis revealed that the OsPDCD5 protein was widely expressed in the tassel, leaf, leaf sheath, and different parts of the stem but not in the anther. RT-PCR analysis showed that OsPDCD5 was related to the senescence of leaf and root tissues as well as the development of stem tissues. Furthermore, OsPDCD5 was up-regulated by UV-B irradiation. Calcineurin B-like interacting protein kinase 23 (OsCIPK23), which is involved in the calcineurin B-like proteins (CLBs)/CBL-interacting protein kinases (CIPKs) signaling network, was identified as interacting with OsPDCD5 by yeast two-hybrid screening and subsequently confirmed by pull-down assay in vitro. Present findings may shed light on the investigation of the biochemical function of OsPDCD5 in rice.