Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (λ(max) = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (λ(max) = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ(max) = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, λ(max) = 595 nm) reverts very rapidly (τ ~ 20 s) back to the thermostable Z state (λ(max) = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1/E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1/Z-GAF3 has intermediate activity, and Z-GAF1/Z-GAF3 is the least active state.     

八角茴香春果和秋果以及不同部位的莽草酸含量分析

采用HPLC法对不同季节和不同部位的八角茴香药材主成分莽草酸的含量进行分析,发现枝条、叶片、春果、秋果的莽草酸含量依次为2.64%、4.94%、8.63%、12.16%;色谱条件:Venusil HILIC(5μm,4.6 mm×250 mm)色谱柱,流动相:乙腈-0.5%三氟乙酸(95:5),流速1.0 mL·min~(-1),检测波长为210 nm。莽草酸进样量在0.001～6μg范围内线性关系良好。莽草酸的线性回归相关系数r=0.9984,平均回收率为98.4%,相对标准偏差(RSD)为1.26%。该研究为制定八角茴香药材质量检测标准提供了简便、快速、准确的方法。     

Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus

? Brassinosteroids (BRs) are a new class of plant hormones that are essential for plant growth and development. Here, the involvement of BRs in plant systemic tolerance to biotic and abiotic stresses was studied. ? The effects of 24-epibrassinolide (EBR) on plant stress tolerance were studied through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation, the analysis of gene expression using quantitative real-time PCR and the measurement of hydrogen peroxide (H?O?) production using a spectrophotometric assay or confocal laser scanning microscopy. ? Treatment of primary leaves with EBR induced systemic tolerance to photooxidative stress in untreated upper and lower leaves. This was accompanied by the systemic accumulation of H?O? and the systemic induction of genes associated with stress responses. Foliar treatment of EBR also enhanced root resistance to Fusarium wilt pathogen. Pharmacological study showed that EBR-induced systemic tolerance was dependent on local and systemic H?O? accumulation. The expression of BR biosynthetic genes was repressed in EBR-treated leaves, but elevated significantly in untreated systemic leaves. Further analysis indicated that EBR-induced systemic induction of BR biosynthetic genes was mediated by systemically elevated H?O?. ? These results strongly argue that local EBR treatment can activate the continuous production of H?O?, and the autopropagative nature of the reactive oxygen species signal, in turn, mediates EBR-induced systemic tolerance.     

E3 Ubiquitin Ligase CHIP and NBR1-Mediated Selective Autophagy Protect Additively against Proteotoxicity in Plant Stress Responses

Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein''s propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.     

Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO₂ assimilation and quantum yield of PSII (Φ_PSII). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO₂ assimilation and Φ_PSII. Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V _c,max), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J _max), thereby increasing maximum carboxylation rate of Rubisco (V _c,max). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.     

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber ( Cucumis sativus L.) and tomato ( Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate–glutathione (AsA–GSH) cycle enzymes and the production of H₂O₂ in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA–GSH cycle enzymes followed by an increased H₂O₂ accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.     

广西千层塔资源调查研究

通过对广西千层塔主要分布区10个野外样点和总样方量30.5 km~2的调查,结合6个标本馆45份千层塔馆藏标本及现有文献的研究表明:千层塔在广西主要分布在桂东北、桂东南等地,实际分布生境总面积约1886.3 km~2;野外生物量调查得到了千层塔在不同地理区域的生物量分布格局,并结合科学评估方法对样方调查结果和间接推导数据的权重分析及环境参数进行评估,初步确定广西千层塔天然生物量为413.1 t;进一步考虑人为采集能力的前提下,估测广西千层塔的理论可采量20.6 t。千层塔资源分布分散,且主要分布在自然保护区和风景区范围内,因此,不宜大规模开采,应进一步寻找科学合理的途径解决资源紧缺问题。     

A Study on Pollen Morphology of Potamogeton,Zannichellia and Triglochin from China

The pollen morphology of 19 species in the genera Potamogeton, Zannichellia and Triglochin from China was examined under light and scanning electron microscopes. 1. Pollen characteristics (1) Potamogeton L. (Plate 1, 2:1-8) Pollen grains spheroidal, subspheroidal, rarely oblate or ovoid, (17-35)24.41×27.76(18-45)μm in size; anaperturate; exine 1-2μm thick, 2layered or obscure; crass-reticulate, reticulate or finely-reticulate under LM, distinctly reticulate under SEM; muri uniform or not uniform, smooth or granular, rarely with processes; bacula thicker or thinner at the intersection of muri. (2) Zannichellia L. (Plate 2:9) Pollen grains spheroidal, ,(20-26)23.1×24.15(21-27) μm in size; anaperturate; exine 1 μm thick, stratification obscure; nearly reticulate under LM, shallowly reticulate under SEM; luminae shallow, large and irregular, muri very thin and not uniform; bacula absent. (3) Triglochin L. (Plate 2:10-11) Pollen grains spheroidal or subspheroidal; (20-30)23.65×23.7(20-30) μm in size; anaperturate; exine about 1μm thick, stratification obscure, finely reticulate under LM, distinctly reticulate under SEM; luminae rather large or minute; muri uniform, with 1-2 lines of granules, bacula present or absent. 2. Pollen types of Potamogeton L. (1) Thin muri--thick bacula: Potamogeton pectinatus, P. intramongolicus, P. filiformis, P. pamiricus; (2) Uniform muri and bacula: 1) Large pollen subtype: Potamogeton crespus; 2) Medium-sized pollen subtype: Potamogeton heterophyllus, P. perfoliatus, P. distinctus, P. malaianus; 3) Minute pollen subtype: Potamogeton oxyphyllus, P. obtusifolius, P. lucens, P. cristatus, P. gramineus, P. octandrus, P. pusillus     

Cre/LoxP系统在转基因小鼠上的应用策略

Cre/LoxP位点特异重组酶系统已发展为在体内外进行遗传操作的一个新的有力工具.该系统在转基因小鼠上的应用,可使转基因的表达或靶基因的缺失/突变的位点特异DNA重组不仅发生在小鼠发育的某一阶段或特定的组织器官,而且,若与控制Cre表达或功能的诱导系统结合,则可以时空方式体现.这些基于重组的策略可能对基因功能的研究和人类疾病的动物模型的建立产生深刻影响.