Sub-cellular localization of PELPK1 in Arabidopsis thaliana as determined by translational fusion with green fluorescent protein reporter

PELPK1, a novel Arabidopsis thaliana gene was earlier annotated to encode a protein of sub-family, PELPK under hydroxyproline-rich glycoprotein (HRGP) super-family of proteins. Previous bioinformatics and computational analyses predicted PELPK1 to contain an amino-terminal signal peptide destined towards the secretory pathway. In the present study, transgenic plants were developed harboring a translational fusion construct comprising of PELPK1 coding sequence (PELPK1-CDS) and green fluorescent protein (GFP) reporter to determine the localization of PELPK1 in Arabidopsis plants. By employing the techniques of confocal laser scanning microscopy, immunolabeling of GFP with quantum dot (Q-dot), and transmission electron microscopy (TEM), it is shown that the translational fusion product is predominantly deposited to the cell wall. These results are in agreement with the earlier bioinformatics prediction that the PELPK1 is transported via the secretory pathway.     

The AtTudor2, a protein with SN-Tudor domains, is involved in control of seed germination in Arabidopsis

The 4SN-Tudor domain protein is an almost ubiquitous eukaryotic protein with four Staphylococcal nuclease domains at the N terminus and a Tudor domain towards the C terminus. It has been found that Tudor-SN protein has multiple roles in governing gene expression during cell growth and development in animals. In plant, although Tudor-SN orthologs have been found in rice, pea and Arabidopsis, and are associated with cytoskeleton, their roles in growth and development are poorly understood. In this study, we investigated the function of Arabidopsis Tudor-SN protein, AtTudor. Our results indicated that the expression of AtTudor2 in seeds was evidently higher than in other tissues. Furthermore, we found that the expression of a key enzyme for GA biosynthesis, AtGA20ox3, was downregulated obviously in AtTudor2 T-DNA insertion mutant and AtTudor1/AtTudor2 RNAi transgenic lines. Together, our results suggest that AtTudor2 is involved in GA biosynthesis and seed germination of Arabidopsis.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seed‐specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth

Enzymatic and non‐enzymatic antioxidants play important roles in the tolerance of abiotic stress. To increase the resistance of seeds to oxidative stress, At2S3 promoter from Arabidopsis was used to achieve overexpression of the antioxidants in a seed‐specific manner. This promoter was shown to be capable of driving the target gene to have a high level of expression in seed‐related organs, including siliques, mature seeds, and early seedlings, thus making its molecular farming applications in plants possible. Subsequently, genes encoding Mn‐superoxide dismutase (MSD1), catalase (CAT1), and homogentisate phytyltransferase (HPT1, responsible for the first committed reaction in the tocopherol biosynthesis pathway) were overexpressed in Arabidopsis under the control of the At2S3 promoter. Double overexpressers co‐expressing two enzymes and triple overexpressers were produced by cross pollination. Mn‐SOD and total CAT activities, as well as γ‐tocopherol content, significantly increased in the corresponding overproduction lines. Moreover, single MSD1‐transgene, double, and triple overexpressers displayed remarkably enhanced oxidative stress tolerance compared to wild type during seed germination and early seedling growth. Interestingly, an increase in the total CAT activity was also observed in the single MSD1‐transgenic lines as a result of MSD1 overexpression. Together, the combined increase in Mn‐SOD and CAT activities in seeds plays an essential role in the improvement of antioxidant capacity at early developmental stage in Arabidopsis.     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

Arabidopsis thaliana Atrab28: a nuclear targeted protein related to germination and toxic cation tolerance

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues.Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.     

IQM1基因过量表达对拟南芥气孔运动及根系生长的影响

拟南芥钙调素结合蛋白IQM家族共有6个成员,已证实IQM1是一个不依赖Ca2+的钙调素结合蛋白,其功能缺失突变体iqm1表现气孔开度小和根短的表型,而且突变体气孔开度并不因光、暗、脱落酸等诱导而变大或变小。该实验构建了IQM1基因双元表达载体并转化拟南芥,通过分子筛选及IQM1表达量分析,获得了IQM1基因过量表达植株。表型分析发现,IQM1过量表达植株在光诱导气孔开放处理后气孔开放度明显比野生型和iqm1-1增大,在暗诱导气孔关闭处理后气孔开度则显著变小;IQM1过量表达植株的主根比野生型和iqm1-1长,侧根数量比野生型和iqm1-1多,但IQM1过量表达对植株的生长形态、抽薹期、开花期及座果等方面却没有明显影响。研究表明,IQM1基因在植物气孔运动及根系生长中起着重要作用。     

Overexpression of the AtSTK gene increases salt, PEG and ABA tolerance in Arabidopsis

AtSTK (At5g02800), which is a serine-threonine protein kinase gene of Arabidopsis thaliana, was cloned, and its function was studied. The study found that the overexpression of AtSTK could significantly improve the ability of A. thaliana to tolerate salt, PEG, and ABA stresses. RT-PCR analysis revealed that the expression of the AtSTK gene could be obviously induced by salt, PEG, and ABA. The examination of the physiological characteristics showed that the overexpression of AtSTK in Arabidopsis significantly reduced the plasma membrane permeability, significantly increased the proline content, and decreased the MDA content. These changes may reflect the physiological mechanisms through which AtSTK overexpression improves stress resistance in Arabidopsis. In addition, the overexpression of the AtSTK gene significantly antagonised the inhibitory effect of high concentrations of exogenous ABA on Arabidopsis seed germination. The subcellular localisation results showed that AtSTK is located in both the cytosol and the nucleus. The examination of its tissue-specific expression showed that AtSTK is expressed in various Arabidopsis tissues and is particularly strongly expressed in the vessels. The signalling pathway analysis indicated that AtSTK might transfer the salt stress signal in Arabidopsis through the MAPK pathway.     

AtNHX3 is a vacuolar K+/H+ antiporter required for low‐potassium tolerance in Arabidopsis thaliana

Three vacuolar cation/H⁺ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na⁺/H⁺ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T‐DNA insertion mutant and 35S‐driven AtNHX3 over‐expression Arabidopsis plants. RT‐PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre‐vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K⁺‐deficient conditions. Expression of AtNHX3 complemented the sensitivity to K⁺ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over‐expressing AtNHX3 displayed significantly higher K⁺/H⁺ exchange rates than those isolated from wild‐type plants. Furthermore, nhx3 seeds accumulated less K⁺ and more Na⁺ when both wild‐type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K⁺/H⁺ antiporter required for low‐potassium tolerance during germination and early seedling development, and may function in K⁺ utilization and ion homeostasis in Arabidopsis.     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

Altered expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO₂ assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes. These authors contributed equally to this study.     

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.