RNA干涉AtSUS3影响拟南芥SUS家族表达模式及角果成熟

蔗糖合成酶（SuSy）是植物蔗糖代谢的关键酶,在植物生长发育过程中起着重要作用.为研究拟南芥中SUS3的功能,构建RNAi-SUS3干涉载体,通过农杆菌介导的真空渗透法转化拟南芥.筛选获得纯系转基因植株后,对AtSUS家族进行表达分析,利用环境扫描电子显微镜观察转基因植株表型,并对转基因拟南芥角果进行木质素组织化学染色以及透射电子显微镜检测.结果表明,RNA干涉技术能够抑制AtSUS3的表达,正常培养条件下该基因沉默后对拟南芥的表型没有显著影响,但可引起角果中AtSUS1,AtSUS2和AtSUS4表达代偿性增加,使转基因植株角果内果皮层细胞次生细胞壁增厚,木质化程度加深,同时果瓣厚度也有增加趋势.结果提示,转基因拟南芥角果的发育较野生型植株更为优先,AtSUS3基因沉默可能有利于角果的成熟.     

Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.     

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.     

DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Background

The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking.

Findings

We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures.

Conclusion

Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2.     

Diphtheria toxin-mediated cell ablation reveals interregional communication during Arabidopsis seed development

Fertilization of the female gametophyte in angiosperm plants initiates a process of coordinated development of embryo, endosperm, and seed coat that ensures the production of a viable seed. Mutant analysis has suggested that communication between the endosperm and the seed coat is an important determinant in this process. In addition, cell groups within the embryo, derived from the apical and from the basal cell, respectively, after zygote division, concertedly establish a functional root meristem, and cells in the apical region of the embryo are hypothesized to repress cell divisions in the basal cell-derived suspensor. The available evidence for these interregional communication events mostly relies on the analysis of mutant phenotypes in Arabidopsis. To provide independent and direct evidence for communication events, we used conditional domain-specific expression of the diphtheria toxin A chain (DTA) in developing Arabidopsis seeds. By using a collection of cell- or tissue-type-specific promoters, we show that the mGAL4:VP16/UAS two-component gene expression allows reliable spatiotemporal and conditional expression of the GFP:GUS reporter and the DTA gene in the developing embryo and endosperm. Expression of DTA in the protoderm of the embryo proper led to excessive proliferation of suspensor cells, sometimes resulting in the formation of secondary embryos. Endosperm-specific expression of DTA caused complete cessation of seed growth, followed by pattern defects in the embryo and embryo arrest. Taken together, the results presented here substantiate the evidence for and underline the importance of interregional communication in embryo and seed development and demonstrate the usefulness of conditional toxin expression as a method complementary to phenotypic analysis of developmental mutants.     

Natural variation in the degree of autonomous endosperm formation reveals independence and constraints of embryo growth during seed development in Arabidopsis thaliana

Seed development in flowering plants is a paradigm for the coordination of different tissues during organ growth. It requires a tight interplay between the two typically sexually produced structures: the embryo, developing from the fertilized egg cell, and the endosperm, originating from a fertilized central cell, along with the surrounding maternal tissues. Little is known about the presumptive signal transduction pathways administering and coordinating these different tissues during seed growth and development. Recently, a new signal has been identified emanating from the fertilization of the egg cell that triggers central cell proliferation without prior fertilization. Here, we demonstrate that there exists a large natural genetic variation with respect to the outcome of this signaling process in the model plant Arabidopsis thaliana. By using a recombinant inbred line population between the two Arabidopsis accessions Bayreuth-0 and Shahdara, we have identified two genetic components that influence the development of unfertilized endosperm. Exploiting this natural variation, we could further dissect the interdependence of embryo and endosperm growth during early seed development. Our data show an unexpectedly large degree of independence in embryo growth, but also reveal the embryo's developmental restrictions with respect to endosperm size. This work provides a genetic framework for dissection of the interplay between embryo and endosperm during seed growth in plants.     

Control of seed development in Arabidopsis thaliana by atmospheric oxygen

Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5·1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2 and 21·3 kPa O₂, 0·035 kPa CO₂ and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O₂ treatments below 16·2 kPa, and seeds from plants grown in 2·5 kPa O₂ did not germinate at all. Fewer than 25% of the seeds from plants grown in 5·1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5·1 kPa O₂, at around the curled cotyledon stage in 10·1 kPa O₂, and at the premature stage in 16·2 kPa O₂. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2·5 kPa O₂. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis .     

Arabidopsis thaliana Oxa proteins locate to mitochondria and fulfill essential roles during embryo development

Members of the Alb3/Oxa1/YidC protein family function as insertases in chloroplasts, mitochondria, and bacteria. Due to independent gene duplications, all organisms possess two isoforms, Oxa1 and Oxa2 except gram-negative bacteria, which encode only for one YidC-like protein. The genome of Arabidopsis thaliana however, encodes for eight different isoforms. The localization of three of these isoforms has been identified earlier: Alb3 and Alb4 located in thylakoid membranes of chloroplasts while AtOxa1 was found in the inner membrane of mitochondria. Here, we show that the second Oxa1 protein, Oxa1b as well as two Oxa2 proteins are also localized in mitochondria. The last two isoforms most likely encode truncated versions of Oxa-like proteins, which might be inoperable pseudogenes. Homozygous mutant lines were only obtained for Oxa1b, which did not reveal any significant phenotypes, while T-DNA insertion lines of Oxa1a, Oxa2a and Oxa2b resulted only in heterozygous plants indicating that these genes are indispensable for plant development. Phenotyping heterozygous lines showed that embryos are either retarded in growth, display an albino phenotype or embryo formation was entirely abolished suggesting that Oxa1a and both Oxa2 proteins function in embryo formation although at different developmental stages as indicated by the various phenotypes observed.     

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.     

Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana

Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.     

The roles of autophagy in development and stress responses in Arabidopsis thaliana

Autophagy is a dynamic process that involves the recycling process of the degradation of intracellular materials. Over the past decade, our molecular and physiological understanding of plant autophagy has greatly been increased. Most essential autophagic machineries are conserved from yeast to plants. The roles that autophagy-related genes (ATGs) family play in the lifecycle of the Arabidopsis are proved to be similar to that in mammal. Autophagy is activated during certain stages of development, senescence or in response to starvation, or environmental stress in Arabidopsis. In the progression of autophagy, ATGs act as central signaling regulators and could develop sophisticated mechanisms to survive when plants are suffering unfavorable environments. It will facilitate further understanding of the molecular mechanisms of autophagy in plant. In this review, we will discuss recent advances in our understanding of autophagy in Arabidopsis, areas of controversy, and highlight potential future directions in autophagy research.     

Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.     

Spectral quality influence of light during development of Arabidopsis thaliana plants in regulating seed germination

During development of Arabidopsis thaliana plants, the spectralquality of light can influence the phytochrome controlled germinationof the progeny. Plants grown under light rich in far red energiesproduce light requiring seed that contain phytochrome in thered absorbing or inactive form. Plants grown under light deficientin far red energies produce dark germinating seed that containphytochrome converted mainly to the far red absorbing or activeform. These responses are determined locally within the developingseed without influence from the vegetative portion of the plant.The developing seed is sensitive to spectral changes throughoutembryogenesis until the seed begins to dehydrate just beforefull maturation. At that point the phytochrome is stabilizedin the form photoinduced before dehydration. In relation tothe induction of phytochrome controlled germination responses,the developing seed appears to act independently of the parentplant. (Received April 16, 1974; )     

The function of SULTR2;1 sulfate transporter during seed development in Arabidopsis thaliana

SULTR2;1 is a low-affinity sulfate transporter expressed in the vascular tissues of roots and leaves for interorgan transport of sulfate in Arabidopsis thaliana . Transgenic Arabidopsis carrying a fusion gene construct of SULTR2;1 5'-promoter region and β-glucuronidase coding sequence (GUS) demonstrated that within the reproductive tissues, SULTR2;1 is specifically expressed in the bases and veins of siliques and in the funiculus, which connects the seeds and the silique. The antisense suppression of SULTR2;1 mRNA caused decrease of sulfate contents in seeds and of thiol contents both in seeds and leaves, as compared with the wildtype (WT). The effect of antisense suppression of SULTR2;1 on seed sulfur status was determined by introducing a sulfur-indicator construct, p35S::β_SRx3:GUS, which drives the expression of GUS reporter under a chimeric cauliflower mosaic virus 35S promoter containing a triplicate repeat of sulfur-responsive promoter region of soybean β-conglycinin β subunit (β_SRx3). The mature seeds of F1 plants carrying both the SULTR2;1 antisense and p35S::β_SRx3:GUS constructs exhibited significant accumulation of GUS activities on sulfur deficiency, as compared with those carrying only the p35S::β_SRx3:GUS construct in the WT background. These results suggested that SULTR2;1 is involved in controlling translocation of sulfate into developing siliques and may modulate the sulfur status of seeds in A. thaliana .     

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.     

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.     

Localization of sucrose synthase activity in developing maize kernels by in situ enzyme histochemistry

Sucrose synthase is usually localized by immunocytochemistry, but this method does not show the actual activity of the localized enzyme. A histochemical assay is presented here showing the activity of sucrose synthase by tetrazolium salt precipitation on sections of developing maize kernels. The advantages of the assay are a high sensitivity for low amounts of active sucrose synthase and the independence of specific antibodies.In this study the activity of endosperm sucrose synthase is shown to move gradually from the apical part of the endosperm to the basal endosperm during kernel development. This shift in sucrose synthase activity correlates well with the localization of starch synthesis during kernel development. The assay also shows the early loss of activity in the aleurone layer bordering the embryo, and a loss of activity in the apical aleurone during the final stage of kernel development while the enzyme was still found by immunocytochemistry. This is in contrast to a high sucrose synthase activity in the epithelium of the scutellum, where hardly any labelling was found with antibodies against maize sucrose synthase. Low sucrose synthase activities were found in the pericarp and pedicel parenchyma.Possible functions of the high and low activity patterns in the developing maize kernels and differences between the enzyme assay and immunocytochemistry are discussed.     

Parent-of-origin effects on seed development in Arabidopsis thaliana require DNA methylation

Some genes in mammals and flowering plants are subject to parental imprinting, a process by which differential epigenetic marks are imposed on male and female gametes so that one set of alleles is silenced on chromosomes contributed by the mother while another is silenced on paternal chromosomes. Therefore, each genome contributes a different set of active alleles to the offspring, which develop abnormally if the parental genome balance is disturbed. In Arabidopsis, seeds inheriting extra maternal genomes show distinctive phenotypes such as low weight and inhibition of mitosis in the endosperm, while extra paternal genomes result in reciprocal phenotypes such as high weight and endosperm overproliferation. DNA methylation is known to be an essential component of the parental imprinting mechanism in mammals, but there is less evidence for this in plants. For the present study, seed development was examined in crosses using a transgenic Arabidopsis line with reduced DNA methylation. Crosses between hypomethylated and wild-type diploid plants produced similar seed phenotypes to crosses between plants with normal methylation but different ploidies. This is consistent with a model in which hypomethylation of one parental genome prevents silencing of alleles that would normally be active only when inherited from the other parent - thus phenocopying the effects of extra genomes. These results suggest an important role for methylation in parent-of-origin effects, and by inference parental imprinting, in plants. The phenotype of biparentally hypomethylated seeds is less extreme than the reciprocal phenotypes of uniparentally hypomethylated seeds. The observation that development is less severely affected if gametes of both sexes (rather than just one) are 'neutralized' with respect to parent-of-origin effects supports the hypothesis that parental imprinting is not necessary to regulate development.