Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation.Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1.Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 ((C)o-(r)egulated with (N)YE1).Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios,which is comparable with that in acd1-20,but much more severe than that in nye1-1.Notably,various Chl binding proteins,especially trimeric LHCP Ⅱ,were markedly retained in crn1-1 four days after dark-treatment,possibly due to a lesion in disassociation of protein-pigment complex.Nevertheless,the photochemical efficiency of PSII in crn1-1 declined,even more rapidly,two days after dark-treatment,compared to those in Col-0 and nye1-1.Our results suggest that CRN1 plays a crucial role in Chl degradation,and that loss of its function produces various side-effects,including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.     

Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10¹ and 10⁵ cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Regulation of Leaf Senescence and Crop Genetic Improvement

Leaf senescence can impact crop production by either changing photosynthesis duration,or by modifying the nutrient remobilization efficiency and harvest index.The doubling of the grain yield in major cereals in the last 50 years was primarily achieved through the extension of photosynthesis duration and the increase in crop biomass partitioning,two things that are intrinsically coupled with leaf senescence.In this review,we consider the functionality of a leaf as a function of leaf age,and divide a leaf’s life into three phases:the functionality increasing phase at the early growth stage,the full functionality phase,and the senescence and functionality decreasing phase.A genetic framework is proposed to describe gene actions at various checkpoints to regulate leaf development and senescence.Four categories of genes contribute to crop production:those which regulate (I) the speed and transition of early leaf growth,(II) photosynthesis rate,(III) the onset and (IV) the progression of leaf senescence.Current advances in isolating and characterizing senescence regulatory genes are discussed in the leaf aging and crop production context.We argue that the breeding of crops with leaf senescence ideotypes should be an essential part of further crop genetic improvement.     

QTL analysis of pest resistance in the wild tomato Lycopersicon pennellii: QTLs controlling acylsugar level and composition

Some accessions of Lycopersicon pennellii, a wild relative of the tomato Lycopersicon esculentum, are resistant to a number of important pests of cultivated tomato due to the accumulation of acylsugars, which constitute 90% of the exudate of type-IV trichomes in L. pennellii LA716. An interspecific F₂ population, created by the cross L. esculentum x L. pennellii LA 716, was surveyed for acylsugar accumulation and subjected to RFLP/QTL analysis to determine the genomic regions associated with the accumulation of acylglucoses, acylsucroses, and total acylsugars, as well as with acylglucoses as a percentage of total acylsugars (mole percent acylglucoses). Data were analyzed using MAPMAKER/QTL with and without a log₁₀ transformation. A threshold value of 2.4 (default value for MAPMAKER/QTL) was used, as well as 95% empirically derived threshold values. Five genomic regions, two on chromosome 2 and one each on chromosomes 3, 4 and 11, were detected as being associated with one or more aspects of acylsugar production. The L. esculentum allele is partially dominant to the L. pennellii allele in the regions on chromosomes 2 and 11, but the L. pennellii allele is dominant in the region on chromosome 3. Throughout this study, we report the comparative effects of analytical methodology on the identification of acylsugar QTLs. Similarities between our results and published results for the genus Solanum are also discussed.R. W. Doerge · S.-C. Liu · J. P. Kuai contributed equally to the paper, and we ordered randomly     

Determination of Hepatocellular Carcinoma and Characterization of Hepatic Focal Lesions with Adaptive Multi-Exponential Intravoxel Incoherent Motion Model

PURPOSE: To distinguish hepatocellular carcinoma (HCC) from other types of hepatic lesions with the adaptive multi-exponential IVIM model. METHODS: 94 hepatic focal lesions, including 38 HCC, 16 metastasis, 12 focal nodular hyperplasia, 13 cholangiocarcinoma, and 15 hemangioma, were examined in this study. Diffusion-weighted images were acquired with 13 b values (b?=?0, 3, …, 500 s/mm²) to measure the adaptive multi-exponential IVIM parameters, namely, pure diffusion coefficient (D), diffusion fraction (f_d), pseudo-diffusion coefficient (D_i*) and perfusion-related diffusion fraction (f_i) of the ith perfusion component. Comparison of the parameters of and their diagnostic performance was determined using Mann-Whitney U test, independent-sample t test, one-way analysis of variance, Z test and receiver-operating characteristic analysis. RESULTS: D, D₁* and D₂* presented significantly difference between HCCs and other hepatic lesions, whereas f_d, f₁ and f₂ did not show statistical differences. In the differential diagnosis of HCCs from other hepatic lesions, D₂* (AUC, 0.927) provided best diagnostic performance among all parameters. Additionally, the number of exponential terms in the model was also an important indicator for distinguishing HCCs from other hepatic lesions. In the benign and malignant analysis, D gave the greatest AUC values, 0.895 or 0.853, for differentiation between malignant and benign lesions with three or two exponential terms. Most parameters were not significantly different between hypovascular and hypervascular lesions. For multiple comparisons, significant differences of D, D₁* or D₂* were found between certain lesion types. CONCLUSION: The adaptive multi-exponential IVIM model was useful and reliable to distinguish HCC from other hepatic lesions.     

Regulation of Triacylglucose Fatty Acid Composition (Uridine Diphosphate Glucose:Fatty Acid Glucosyltransferases with Overlapping Chain-Length Specificity)

UDP-glucose (UDP-Glc):fatty acid glucosyltransferases catalyze the UDP-Glc-dependent activation of fatty acids as 1-O-acyl-[beta]-glucoses. 1-O-Acyl-[beta]-glucoses act as acyl donors in the biosynthesis of 2,3,4-tri-O-acylglucoses secreted by wild tomato (Lycopersicon pennellii) glandular trichomes. The acyl composition of L. pennellii 2,3,4-tri-O-acylglucoses is dominated by branched short-chain acids (4:0 and 5:0; approximately 65%) and straight and branched medium-chain-length fatty acids (10:0 and 12:0; approximately 35%). Two operationally soluble UDP-Glc:fatty acid glucosyltransferases (I and II) were separated and partially purified from L. pennellii (LA1376) leaves by polyethylene glycol precipitation followed by DEAE-Sepharose and Cibacron Blue 3GA-agarose chromatography. Whereas both transferases possessed similar affinity for UDP-Glc, glucosyltransferase I showed higher specificity toward short-chain fatty acids (4:0) and glucosyltransferase II showed higher specificity toward medium-chain fatty acids (8:0 and 12:0). The overlapping specificity of UDP-Glc:fatty acid glucosyltransferases for 4:0 to 12:0 fatty acid chain lengths suggests that the mechanism of 6:0 to 9:0 exclusion from acyl substituents of 2,3,4-tri-O-acylglucoses is unlikely to be controlled at the level of fatty acid activation. UDP-Glc:fatty acid glucosyltransferases are also present in cultivated tomato (Lycopersicon esculentum), and activities toward 4:0, 8:0, and 12:0 fatty acids do not appear to be primarily epidermal when assayed in interspecific periclinal chimeras.     

棒杆菌启动子片段的酶谱分析及定位

用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。     

AtCLH2, a Typical but Possibly Distinctive Chiorophyllase Gene in Arabidopsis

Chlorophyllase (EC 3.1.1.14) is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thallana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here,we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly,"activation" of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.