The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

 Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. Received: 2 June 1999 / Accepted: 13 August 1999     

肠道病毒71型和柯萨奇病毒A16型多重反转录聚合酶链反应的建立及初步应用

本文旨在建立一种快速、高效的方法检测肠道病毒71型（EV71）和柯萨奇病毒A16型(CA16)的方法,以用于儿童手足口病的病原学监测。通过设计肠道病毒通用引物和CA16与EV71的型特异性引物,建立不同引物浓度配比及两阶段退火温度以提高检测敏感性和特异性的多重反转录聚合酶链反应（RT-PCR）方法,并对首都儿科研究所附属儿童医院2010年3~10月收集的371例手足口病患儿共381份临床标本同时进行病毒分离和核酸检测。结果显示,本研究建立的多重RT-PCR方法对CA16和EV71的最低模板检测浓度分别为5.32 pg/ml和0.64 pg/ml,反应特异度为100%。应用该方法检测381份手足口病临床标本的总阳性率为78.4%,其中CA16与EV71的检测阳性率分别为32.6%和35.8%,二者检测阳性比为1:1.1。以病毒分离为标准,多重RT-PCR对CA16及EV71检测的准确率分别为95.2%和98.6%。因此,本研究新建立的多重RT-PCR方法准确、简便,适用于较大量样本的手足口病病原学监测。2010年引起北京地区儿童手足口病的主要病原为CA16和EV71。     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

 The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (β-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUSArabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression. Received: 23 May 1999 / Accepted: 3 December 1999     

Light quantity controls leaf-cell and chloroplast development in Arabidopsis thaliana wild type and blue-light-perception mutants

Plants acclimate to changes in light quantity by altering leaf-cell development and the accumulation of chloroplast components, such that light absorption is favoured under limiting illumination, and light utilisation under non-limiting conditions. Previous evidence suggests an involvement of a high-light photosynthetic redox signal in the down-regulation of the accumulation of the light-harvesting complexes of photosystem II (Lhcb) and the expression of the Lhcb genes, and of a blue-light signal in the control of leaf development and in the increase in photosynthetic capacity, as affected by the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We examined the internal anatomy of leaves, the ultrastructure of chloroplasts and accumulation of light-harvesting complexes and Rubisco in wild-type Arabidopsis thaliana (L.) Heynh. and in mutants in each of the three known blue-light photoreceptors, cryptochrome 1, cryptochrome 2 and phototropin, as well as a mutant in both cryptochromes. Our results indicate an extensive capacity of the Arabidopsis mesophyll cells to adapt to high light fluence rate with an increase in palisade elongation. Under high light, chloroplasts showed increased starch accumulation and reductions in the amount of granal thylakoids per chloroplast, in the proportion of chlorophyll b relative to chlorophyll a, and in the accumulation of the major Lhcb polypeptides. The responses were similar for all four mutants, with respect to their wild types. The results are consistent with either a complete redundancy in function between cryptochromes and phototropin, or their absence of involvement in the light-quantity responses tested. We observed minimal effects of light quantity on Rubisco accumulation over the range of fluence rates used, and conclude that elongation of palisade mesophyll cells and accumulation of Rubisco are controlled separately. This indicates that light acclimation must be the result of a number of individual elementary responses. Quantitative differences in the acclimatory responses were observed between the Landsberg erecta and Columbia wild-type ecotypes used. Received: 4 April 2000 / Accepted: 14 July 2000     

Heteroblasty in Arabidopsis thaliana (L.) Heynh

 Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific β-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emf1lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position. Received: 9 July 1999 / Accepted: 17 August 1999     

The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana

 Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N-terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis. Received: 26 August 1999 / Accepted: 4 October 1999     

A Brassica napus skp1-like gene promoter drives GUS expression in Arabidopsis thaliana male and female gametophytes

We isolated a gene, BnSKP1γ1, expressed in rapeseed (Brassica napus) microspores, which encodes a protein closely related to the Saccharomyces cerevisiae Skp1p protein previously shown to play a role in cell cycle regulation. Twelve SKP1-related genes have already been identified in the Arabidopsis thaliana genome. Using a PCR-based strategy, we isolated three other genes. To date, most data available concerning the function of the SKP1-related genes in plants are indirect. Studies on transgenic A. thaliana plants showthat a 1100-bp BnSKP1γ1 promoter fragment can direct GUS expression in female gametophytes soon after the first haploid mitosis and in male gametophytes from the tetrade stage. No GUS expression can be detected in sporophytic tissues. RT-PCR experiments suggest that this gene is expressed in a similar way in rapeseed. This is the first reported case of a gene exhibiting such an expression pattern in angiosperms. Received: 5 October 1999 / Revision accepted: 28 March 2000     

多重聚合酶链反应与荚膜肿胀试验对肺炎链球菌血清分型的比较研究

为评估多重聚合酶链反应（PCR ）对肺炎链球菌血清分型的可行性,分别采用多重PCR和荚膜肿胀试验对568株肺炎链球菌进行血清分型,并对分型结果进行比较分析。结果显示,568株肺炎链球菌中,213株通过荚膜肿胀试验分出16个血清群,主要有血清群19（23．1％,131／568）、6（5．3％,30／568）、23（1．6％,9／568）、14（1．4％,8／568）、9（1．1％,6／568）、15（1．1％,6／568）等,分型率为37．5％（213／568）;356株通过多重PCR分出21个血清群,主要有血清群19（27．8％,158／568）、23（8．5％,48／568）、6（7．4％,42／568）、14（4．4％,25／568）、3（4．2％,24／568）、15（3．5％,20／568）等,分型率为62．7％（356／568）。荚膜肿胀试验鉴定出血清群4和18,但多重PCR未能鉴定;多重PCR鉴定出血清群5、12、35、16、17和22,但荚膜肿胀试验未能鉴定。多重PCR与荚膜肿胀试验对19F、19A血清型的鉴定无显著差异。结果提示,这2种方法对肺炎链球菌血清分型结果有差别,多重PCR的分型率高于荚膜肿胀试验。对来源复杂的标本进行肺炎链球菌血清分型,2种方法可相互补充,以提高分型率。     

马铃薯病毒一步法多重RT-PCR检测技术的构建

根据马铃薯病毒PVX、PVY、PVA、PLRV的CP基因序列设计4对特异性引物,通过对试剂浓度和反应条件进行优化,建立了能够同步检测PVX、PVY、PVA、PLRV的一步法多重RT-PCR检测方法。该方法对PVX、PVY、PVA、PLRV扩增出的靶带大小分别为732、422、132和336 bp,凝胶电泳易辨别区分。病毒RNA最低检测限度为7.8 pg/μL,对PVM、PVS、AMV、TMV及PSTVd的扩增为阴性。研究结果表明,该方法特异、灵敏,比两步法多重RT-PCR检测更加快速、简便,提高了检测效率,降低检测成本,为马铃薯病毒的高效检测提供了有效手段。     

Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction

AIMS: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. METHODS AND RESULTS: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65.9%) of 91 samples in contrast to 62 (68.12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. CONCLUSIONS: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.     

Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples

PCR assays were formatted using primer pairs homologous to phoE and invA genes. The amplification conditions were optimized with pure cultures and reactions were carried out to define selectivity, specificity and sensitivity of both primer pairs. The performance of the invA primer pair was better than that of the phoE pair, making the specific detection of Salmonella serovars and strains isolated from different food samples possible. Using the invA primer pair, the combined selective enrichment method with the polymerase chain reaction assay was established and used to detect Salmonella from artificially multi-contaminated food samples. The complete procedure detected as few as three cells of Salmonella (3 c.f.u.) from milk and meat samples.     

A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity

Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed to characterize one line (denoted Lsr1⁻) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin, and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished suppression of minimum fluorescence yield (F _o ) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination. Received: 26 May 1999 / Accepted: 17 June 1999     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000