SRK-SCR转基因拟南芥自交不亲和性的研究进展

十字花科植物自交不亲和性（SI）受墨位点（S-locus）编码的sRK和sCR控制,它们分别是柱头和花粉中的sI特异识别因子。野生型拟南芥不具有sI,而近来通过转基因手段将外源艘K—scR基因转入野生型拟南芥可以使其表现sI,由此建立了一个可用于十字花科sI研究的新型模式植物。本文综述了利用这种转基因拟南芥在SI机制及进化方面取得的进展,包括sI新基因的挖掘、候选基因功能分析和拟南芥生殖模式的转变等。     

Biphasic fluence-response curves for light induced germination of Arabidopsis thaliana seeds

Abstract With appropriate pretreatment of the seeds fluence-response curves for the induction of germination of Arabidopsis thaliana show two phases. A proportion of the population responds to very low fluence (VLFR), 10⁴–10^?2μmolm^?2 establishing 10^?4–10^?2% of the total phytochrome in the far-red absorbing form (Pfr) and a proportion of the population respond to low fluence (LFR), 1–1000 μmolm^?2, establishing 1–75% Pfr. The VLFR is nol normally seen because the pre-existing Pfr level satisfies the Pfr requirement or use of green safelight establishes more Pfr than necessary to saturate the VLFR. Endogenous Pfr was depicted by a 24 h 35°C treatment, presumably as a result of dark destruction and/or dark reversion to the red absorbing form of phytochrome (Pr), making it possible to visualize the VLFR. A short pulse of 35°C treatment in combination with an appropriate temperature regime is also able to sensitize a proportion of the seed population. The proportion of the population showing the VLFR is determined by the duration of the cold imbibition pretreatment as well as the duration of the 35°C treatment. Complex fluence-response curves were observed in which a proportion of the seeds being promoted in the VLFR range, were inhibited at higher fluences before being further promoted in the LFR range. This was particularly clear for seed batches being sensitized by a short 35°C treatment. The VLFR may be of significance in the natural environment, enabling seeds buried in the upper layer of the soil to germinate, where the fluence rate falls off sharply and the LFR is not satisfied. A model is presented to explain the two phases in the fluence-response curves.     

Effects of preconditioning and temperature during germination of 73 natural accessions of Arabidopsis thaliana

BACKGROUND AND AIMS: Germination and establishment of seeds are complex traits affected by a wide range of internal and external influences. The effects of parental temperature preconditioning and temperature during germination on germination and establishment of Arabidopsis thaliana were examined. METHODS: Seeds from parental plants grown at 14 and at 22 degrees C were screened for germination (protrusion of radicle) and establishment (greening of cotyledons) at three different temperatures (10, 18 and 26 degrees C). Seventy-three accessions from across the entire distribution range of A. thaliana were included. KEY RESULTS: Multifactorial analyses of variances revealed significant differences in the effects of genotypes, preconditioning, temperature treatment, and their interactions on duration of germination and establishment. Reaction norms showed an enormous range of plasticity among the preconditioning and different germination temperatures. Correlations of percentage total germination and establishment after 38 d with the geographical origin of accessions were only significant for 14 degrees C preconditioning but not for 22 degrees C preconditioning. Correlations with temperature and precipitation on the origin of the accessions were mainly found at the lower germination temperatures (10 and 18 degrees C) and were absent at higher germination temperatures (26 degrees C). CONCLUSIONS: Overall, the data show huge variation of germination and establishment among natural accessions of A. thaliana and might serve as a valuable source for further germination and plasticity studies.     

AtAPOSTART1, an Arabidopsis thaliana PH-START domain protein involved in seed germination

The seed in the mature and dry state is metabolically inactive (quiescent) and is thus able to withstand extreme environmental conditions, such as drought and cold. Germination commences when the dry seed, shed from its parent plant, takes up water (imbibition) and ends when the root emerges through the seed coat. During seedling establishment, the reserves stored in the seed are metabolized, whereas the subsequent vegetative and reproductive growth is supported by photosynthesis. Here, we describe the functional characterization of the PH-START protein AtAPO1 (Arabidopsis thaliana APOSTART1), the putative homologue of PpAPO1 (Poa pratensis APOSTART1) in Arabidopsis thaliana. By using translational fusion of the AtAPO1 promoter to the uiaD gene and in situ hybridization analyses, we show that AtAPO1 is expressed in mature embryo sacs and developing embryos. The functional analysis of two at-apostart mutant alleles suggests that AtAPO1 is involved in the control of seed germination.     

Arabidopsis thaliana seed germination and early seedling growth are inhibited by monoethanolamine salts of para-halogenated benzoic acids

Abstract

In spite of the simplicity of its molecules, the complex effects of benzoic acids on the regulation of plant growth are an increasingly attractive field of research to chemists and biologists. Halide substituted benzoic acids offer an excellent opportunity to explore the effect of electron withdrawing substituents (fluoro-, chloro-, bromo- and iodo-) on the response of plant growth stage. Under normal physiological conditions, benzoic acids are ionized molecules that exhibit low solubility in water. Monoethanolamine, a natural alkanolamine, was used to generate salts of monoethanolamine of halogenated para-substituted benzoic acids, new compounds with biological activity. This study reports on the biological effects of these substances at different concentrations on Arabidopsis thaliana seed germination and early seedling growth. Seed germination at 22°C, in a vertical position, under a photoperiod of 16 h light and 8 h darkness, was variable depending on the concentration of the compounds applied. Final germination percentages were similar for all treatments and control at 0.05 mM and 0.1 mM (exception p-Br BA and p-I MEASPBA). No germination occurred when seeds were treated with more than 0.5 mM. The results also revealed that the primary root length and the number of secondary roots are reduced in a concentration-dependent manner and also in relation to increasing atomic size of the substituents (F < Cl < Br < I). It is concluded that uptake rates of benzoic acid anions by roots decrease with a decrease in hydrophilic character of the anion and with an increase in molecular size.     

Changes in the isozyme composition of antioxidant enzymes in response to aminotriazole in leaves ofArabidopsis thaliana

The changes in isozyme profiles of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) during severe deactivation of total CAT activity by aminotriazole (AT) treatment were investigated in the leaves ofArabidopsis thaliana (Columbia ecotype) in relation to H₂O₂-mediated oxidative stress. In spite of striking deactivation of total CAT activity by 0.1 mM AT, there were no significant differences in H₂O₂ levels or total leaf soluble protein contents including a Rubisco in both the control and AT-treated leaves. On the other hand, one specific protein band (molecular mass, 66 kD) was observed on the SDS-gel from leaf soluble proteins whose staining intensity was strikingly enhanced by AT treatment for 6 h. However, this band disappeared at 12 h. In the native-gel assays of CAT, POD, APX and GR isozymes, AT remarkably inhibited the expression of the CAT1 isozyme with no effects on CAT2 and CAT3, and generally had no effect on POD isozyme profiles. However, AT stimulated the intensity of activities of pre-existing APX1 and GR1 isozymes. In particular, it induced a new synthesis of one GR isozyme. Therefore, these results collectively suggest that a striking deactivation of total CAT activity by AT inA. thaliana leaves largely results from the suppression of CAT1 isozyme, and that APX1, GR1, and a newly synthesized GR isozyme could complement the role of CAT1 to metabolize H₂O₂ into non-toxic water.     

重金属镉对拟南芥DNA甲基化的影响

将拟南芥种子点种于添加有不同浓度CdCl2的培养基中处理2周,移苗时CdCl2的胁迫即解除。低浓度CdCl2促进拟南芥种子的萌发。CdCl2为0．5mg·L^-1时萌发率最高（为97．21％）。随着CdCl2浓度的继续增加,种子萌发率即逐渐下降。幼苗期和抽薹期分别提取叶DNA,采用甲基敏感扩增多态性（MSAP）技术分析其基因组DNA甲基化的结果显示,总的来说,随着CdCl2浓度的增加,甲基化程度增高。     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins （2-Cys Prxs） are efficiently reduced by NADPH Thioredoxin reductase C （NTRC）. To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase （GS）, and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wildtype and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.     

Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds

Gibberellin levels in imbibed Arabidopsis thaliana seeds are regulated by light via phytochrome, presumably through regulation of gibberellin biosynthesis genes, AtGA3ox1 and AtGA3ox2, and a deactivation gene, AtGA2ox2. Here, we show that a loss-of-function ga2ox2 mutation causes an increase in GA(4) levels and partly suppresses the germination inability during dark imbibition after inactivation of phytochrome. Experiments using 2,2-dimethylGA(4), a GA(4) analog resistant to gibberellin 2-oxidase, in combination with ga2ox2 mutant seeds suggest that the efficiency of deactivation of exogenous GA(4) by AtGA2ox2 is dependent on light conditions, which partly explains phytochrome-mediated changes in gibberellin effectiveness (sensitivity) found in previous studies.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

新疆北部拟南芥自然居群表型变异与协变

拟南芥(Arabidopsis thaliana)自然居群的表型特征代表其在自然环境下的适应状况, 不同居群间特征的对比可以为了解拟南芥表型变化规律, 进而分析其形成过程和机制提供重要线索。本研究以分布于新疆北部天山、塔尔巴哈台山和阿尔泰山的10个种群的9个表型性状为基础, 对比分析了小尺度、局域尺度和区域尺度环境下原生境拟南芥种群表型性状的变化。结果发现, 不同性状对环境变化的反应不同, 其中株高、株重、根重、根长、单个果实重、果实开裂力度在3种环境尺度下种群间的差异均达到极显著水平, 而分枝数、果实长度的种群间变化不显著, 种群间的表型分化系数较低。不同环境尺度下株重、根重、单株果数均表现出一致的协变格局, 反映了生理功能性状之间整合对拟南芥适应环境的重要性。同时, 各种群间整体的性状协变差异性明显, 根长、单个果实重、分枝数、果实长度、果实开裂力度等特征与其他特征协变具有明显的局部性, 局域尺度和区域尺度环境之间的变化较大。聚类分析发现区域尺度上的不同种群聚合在一起的现象非常突出, 进一步表明拟南芥的表型特征受微环境的强烈影响。Mantel检验表明, 小尺度上10个种群株高、株重、根重、单个果实重、果实长度、果实开裂力度6个性状变化存在显著的空间相关性, 而分枝数、根长的相关性却不显著。因此, 我们认为拟南芥表型变化受小尺度环境的影响强烈, 但在表型层面并非所有性状都与原生境气候存在遗传关联。     

Measuring spatial variation in natural selection using randomly-sown seeds of Arabidopsis thaliana

We describe a simple method of measuring spatial variation in fitness using randomly sown seeds. Given a random initial distribution of seeds, any spatial variation in the final distribution of genotypes must be the result of spatial variation in natural selection. Departures from spatial randomness are tested using a modification of join-count statistics, based on the probability that two randomly chosen plants separated by a given distance carry the same genetic marker. Monte Carlo simulations showed that for the randomly sown seeds design the join-count test had higher power to detect spatial variation in fitness than analysis of variance. The power was relatively independent of the number of genotypes used or the number of microsites sampled. The method was tested in the field using three standard lines of Arabidopsis thaliana. At the end of the life cycle, adjacent plants were significantly more likely to be the same line than randomly chosen pairs and the spatial dependence extended to distances of nearly 50 cm. Simple experiments like this may be especially useful for “comparative ecological genetics”, allowing systematic studies of natural selection among many species or habitats.     

Effects of light and temperature on seed dormancy and gibberellin-stimulated germination in Arabidopsis thaliana: studies with gibberellin-deficient and -insensitive mutants.

Effects of light and temperature on gibberellin (GA)-induced seed germination were studied in Arabidopsis thaliana (L.) Heynh. with the use of GA-deficient ( gal ) mutants, mutants with a strongly reduced sensitivity to GA ( gai ) and with the recombinant gai/gal . Seeds of the gal mutant did not germinate in the absence of exogenous GAs, neither in darkness, nor in light, indicating that GAs are absolutely required for germination of this species. Wild-type and gai seeds did not always require applied GAs in light. The conclusion that light stimulates GA biosynthesis was strengthened by the antagonistic action of tetcyclacis, an inhibitor of GA biosynthesis. In wild-type, gal and gai/gal seeds light lowered the GA requirement, which can be interpreted as an increase in sensitivity to GAs. In gai and gai/gal seeds light became effective only after dormancy was broken by either a chilling treatment of one week or a dry after-ripening period at 2°C during some months. The present genetic and physiological evidence strongly suggests that temperature regulates the responsiveness to light in A. thaliana seeds. The responsiveness increases during dormancy breaking, whereas the opposite occurs during induction of dormancy (8 days at 15°C pre-incubation). Since light stimulates the synthesis of GAs as well as the responsiveness to GAs, temperature-induced changes in dormancy may indirectly change the capacities to synthesize GAs and to respond to GAs. GA sensitivity is also directly controlled by temperature. It is concluded that both GA biosynthesis and sensitivity to GAs are not the primary controlling factors in dormancy, but are essential for germination.     

Inferring the geometry of fourth-period metallic elements in arabidopsis thaliana seeds using synchrotron-based multi-angle X-ray fluorescence mapping

BACKGROUND: Improving our knowledge of plant metal metabolism is facilitated by the use of analytical techniques to map the distribution of elements in tissues. One such technique is X-ray fluorescence (XRF), which has been used previously to map metal distribution in both two and three dimensions. One of the difficulties of mapping metal distribution in two dimensions is that it can be difficult to normalize for tissue thickness. When mapping metal distribution in three dimensions, the time required to collect the data can become a major constraint. In this article a compromise is suggested between two- and three-dimensional mapping using multi-angle XRF imaging. METHODS: A synchrotron-based XRF microprobe was used to map the distribution of K, Ca, Mn, Fe, Ni, Cu and Zn in whole Arabidopsis thaliana seeds. Relative concentrations of each element were determined by measuring fluorescence emitted from a 10 microm excitation beam at 13 keV. XRF spectra were collected from an array of points with 25 or 30 microm steps. Maps were recorded at 0 and 90 degrees , or at 0, 60 and 120 degrees for each seed. Using these data, circular or ellipsoidal cross-sections were modelled, and from these an apparent pathlength for the excitation beam was calculated to normalize the data. Elemental distribution was mapped in seeds from ecotype Columbia-4 plants, as well as the metal accumulation mutants manganese accumulator 1 (man1) and nicotianamine synthetase (nasx). CONCLUSIONS: Multi-angle XRF imaging will be useful for mapping elemental distribution in plant tissues. It offers a compromise between two- and three-dimensional XRF mapping, as far as collection times, image resolution and ease of visualization. It is also complementary to other metal-mapping techniques. Mn, Fe and Cu had tissue-specific accumulation patterns. Metal accumulation patterns were different between seeds of the Col-4, man1 and nasx genotypes.     

Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization

We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags. A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5 and 3 Race. DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming). Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy. 5 sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize. This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.     

一种简易可行的拟南芥水培新方法

在国内外拟南芥水培方法研究基础上,结合实际情况,设计一套适合于实验室的需要,能就地取材自己制作的水培装置.这套装置主要由两部分组成:播放种子的塑料管和作为支撑物的不锈钢丝网,盛溶液的容器.实验证明两个拟南芥品种在这种水培装置中生长良好,且培养的费用低,易于管理等.由于有钢丝网作支撑,拟南芥能够顺利完成整个生长周期并且结实饱满,种子萌发率高.