A Rice Panicle Mutant Created by Transformation with an Antisense cDNA Library

A rice （Oryza sativa L.） mutant displaying defects in panicle development was identified among transformants in a transgenic mutagenlzed experiment using an antlsense cDNA library prepared from young rice panicles. In the mutant, the average splkelet number was reduced to 59.8 compared with 104.3 in wild-type plants. In addition, the seed-setting rate of the mutant was low （39.3%） owing to abnormal female development. Genetic analysis of T1 and T2 progeny showed that the traits segregated In a 3 （mutant） ： 1 （wild type） ratio and the mutation was cosegregated with the transgene. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses showed that the mutant had a single T-DNA insertion on chromosome 5, where no gene was tagged. Sequencing analysis found that the transgenic antisense cDNA was derived from a gene encoding an F-box protein in chromosome 7 with unidentified function. This and another four homologous genes encoding putative F-box proteins form a gene cluster. These results indicate that the phenotyplc mutations were most likely due to the silencing effect of the expressed transgenic antisense construct on the member（s） of the F-box gene cluster.     

Biological and biochemical characterization of a red-eye mutant in Nilaparvata lugens （Hemiptera： Delphacidae）

A red-eye colony was established in our laboratory in brown planthopper （BPH）, Nilaparvata lugens （Stal）, a major rice pest in Asia. Except for the red-eye phenotype, no other differences were observed between the wild-type （brown eye） and the mutant-type （red eye） in external characters. Genetic analysis revealed that the red-eye phenotype was controlled by a single autosomal recessive allele. Biological studies found that egg produc- tion and egg viability in the red-eye mutant colony were not significantly different from those in the wild-type BPH. Biochemical analysis and electronic microscopy examination revealed that the red-eye mutants contained decreased levels of both xanthommatin （brown） and pteridine （red） and reduced number of pigment granules. Thus, the changes of amount and ratio of the two pigments is the biochemical basis of this red-eye mutation. Our results indicate that the red-eye mutant gene （red） might be involved in one common gene locus shared by the two pigments in pigment transportation, pigment granule formation or some other processes.     

Heat Shock Factors HsfB 1 and HsfB2b Are Involved in the Regulation of Pdf1.2 Expression and Pathogen Resistance in Arabidopsis

In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdfl.2a/b in mutant plants. The Pdfexpression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB 1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdfgenes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.     

Redox status of thioredoxin-1 （TRX1） determines the sensitivity of human liver carcinoma cells （HepG2） to arsenic trioxide-induced cell death

lntracellular redox homeostasis plays a critical role in determining tumor cells＇ sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 （TRX1）, a key component of redox regulation, in arsenic trioxide （AS2O3）-induced apoptosis. Over-expression of wild-type TRX1 in HepG2 cells led to the inhibition of As2O3-induced cytochrome c （cyto c） release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG2 cells to As2O3-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys^32/35 to Ser^32/35 converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore （mPTP） opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A （CsA）, indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene （DNCB）, a specific inhibitor ofTRX reductase, also sensitized HepG2 cells to As203-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As2O3-induced apoptosis.     

Arabidopsis PTD Is Required for Type I Crossover Formation and Affects Recombination Frequency in Two Different Chromosomal Regions

In eukaryotes, crossovers together with sister chromatid cohesion maintain physical association between homologous chromosomes, ensuring accurate chromosome segregation during meiosis I and resulting in exchange of genetic information between homologues. The Arabidopsis PTD （Parting Dancers） gene affects the level of meiotic crossover formation, but its functional relationships with other core meiotic genes, such as AtSP011-1, AtRAD51, and AtMSH4, are unclear; whether PTD has other functions in meiosis is also unknown. To further analyze PTD function and to test for epistatic relationships, we compared the meiotic chromosome behaviors ofAtspoll-1 ptd and Atrad51 ptd double mutants with the relevant single mutants. The results suggest that PTD functions downstream of AtSP011-1 and AtRAD51 in the meiotic recombination pathway. Furthermore, we found that meiotic defects in rck pM and Atmsh4 ptd double mutants showed similar meiotic phenotypes to those of the relevant single mutants, providing genetic evidences for roles of PTD and RCK in the type I crossovers pathway. Moreover, we employed a pollen tetrad-based fluorescence method and found that the meiotic crossover frequencies in two genetic intervals were significantly reduced from 6.63% and 22.26% in wild-type to 1.14% and 6.36%, respectively, in the ptd~2 mutant. These results revealed new aspects of PTD function in meiotic crossover formation.     

Arabidopsis tic62 trol Mutant Lacking Thylakoid- Bound Ferredoxin-NADP＋ Oxidoreductase Shows Distinct Metabolic Phenotype

Ferredoxin-NADP＋ oxidoreductase （FNR）, functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP~ was detected. Since the CO2 fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants.     

Acquirement and characterization of a carotenoid mutant (GM309) of Rhodobacter sphaeroides 601

A green mutant was obtained among the chemically induced mutants of Rhodo-bacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular di-chroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.     

OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds

Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium （Cd） in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.     

Rig-I^-/- mice develop colitis associated with downregulation of Gαi2

RIG-I （retinoid acid-inducible gene-I）, a putative RNA helicase with a cytoplasmic caspase-recrultment domain （CARD）, was identified as a pattem-recognition receptor （PRR） that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I^-/- mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I^-/- mice are viable and fertile. Histological analysis shows that Rig-I^-/ mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer＇s patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation ofT-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein αi2 subunit （Gαi2） in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated Gαi2 expression. Moreover, Gαi2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of Gαi2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of Gαi2 and disturbed T-cell homeostasis.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

Characterization and Fine Mapping of a Novel Rice Narrow Leaf Mutant nal9

A narrow leaf mutant was isolated from transgenic rice （Oryza sativa L.） lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 （narrow leaf 9）. The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F2 generations of a cross between the nal9mutant and Longtepu were used for mapping. With 24 F2 mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent CIp protease proteolytic subunit gene （CIpP） was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the CIpP cDNA driven by the 35S promoter. Accordingly, the CIpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.     

PROTEIN S-ACYL TRANSFERASE 13/16 modulate disease resistance by S-acylation of the nucleotide binding,leucine-rich repeat protein R5L1 in Arabidopsis

Nucleotide binding, leucine-rich repeat(NB-LRR)proteins are critical for disease resistance in plants,while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000(Pst DC3000). Of the five mutants that were hyper-...     

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 （wps1）, which shows defects in the development of petals and stamens. Genetic analysis indicates that wpsl mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wpsl mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wpsl mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wpsl flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wpsl acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.     

Prefoldins 3 and 5 Play an Essential Role in Arabidopsis Tolerance to Salt Stress

During the last years, our understanding of the mechanisms that control plant response to salt stress has been steadily progressing. Pharmacological studies have allowed the suggestion that the cytoskeleton may be involved in regulating such a response. Nevertheless, genetic evidence establishing that the cytoskeleton has a role in plant tolerance to salt stress has not been reported yet. Here, we have characterized Arabidopsis T-DNA mutants for genes encoding proteins orthologous to prefoldin （PFD） subunits 3 and 5 from yeast and mammals. In these organisms, PFD subunits, also known as Genes Involved in Microtubule biogenesis （GIM）, form a heterohexameric PFD complex implicated in tubulin and actin folding. We show that, indeed, PFD3 and PFD5 can substitute for the loss of their yeast orthologs, as they are able to complement yeast gim2Δ and gim5Δ mutants, respectively. Our results indicate that pfd3 and pfd5 mutants have reduced levels of α- and β-tubulin compared to the wild-type plants when growing under both control and salt-stress conditions. In addition, pfd3 and pfd5 mutants display alterations in their developmental patterns and microtubule organization, and, more importantly, are hypersensitive to high concentrations of NaCl but not of LiCl or mannitol. These results demonstrate that the cytoskeleton plays an essential role in plant tolerance to salt stress.