Antisense Expression of a Cell Wall–Associated Protein Kinase, WAK4, Inhibits Cell Elongation and Alters Morphology

The Arabidopsis cell wall–associated receptor-like kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the intracellular and extracellular compartments. WAK members are expressed in specific organs and regulated differentially by various biotic and abiotic factors. To gain further insight into how WAKs function during development, we used a glucocorticoid-inducible system to express ectopically the WAK4 antisense gene. The induced expression of the WAK4 antisense gene resulted in a significant decrease of WAK proteins. Ninety-six hours after the induction of WAK4 antisense expression, WAK proteins became undetectable. Cell elongation was impaired, and lateral root development was blocked. The level of WAK protein could be controlled by the concentration of the applied inducer, dexamethasone, and was correlated with the severity of the cell elongation inhibition phenotype. These results suggest that the WAKs serve a vital role in cell elongation and are required for plant development.     

Lens-Specific Expression of PDGF-A Alters Lens Growth and Development

The vertebrate lens provides anin vivomodel to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development byin situhybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-α receptor (PDGF-αR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-αR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens developmentin vivo,we generated transgenic mice that express human PDGF-A in the lens under the control of the αA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific β-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cellsin vivo.Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

GTP-Binding-Defective ARL4D Alters Mitochondrial Morphology and Membrane Potential

ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.     

Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation

We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter. We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism. We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates.     

Aberrant Expression of Basic Fibroblast Growth Factor in NIH-3T3 Cells Alters Drug Resistance and Gene Amplification Potential

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.     

Antisense Expression of an Arabidopsis Ran Binding Protein Renders Transgenic Roots Hypersensitive to Auxin and Alters Auxin-Induced Root Growth and Development by Arresting Mitotic Progress

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.     

反义4CL与UGPase双价基因在烟草中的转化及表达分析

构建了携带紫穗槐尿苷二磷酸葡萄糖焦磷酸化酶（UDP-glucose pyrophosphorylase,UGPase）基因和4-香豆酸：辅酶A连接酶（4-coumarate：coenzyme Aligase,4CL）反义基因的双价基因植物表达载体,用热激法转化至根癌农杆菌LBA4404中,并用制备的农杆菌工程菌进行了烟草转化。PCR及Southern杂交结果证实,双价基因已成功整合到烟草基因组中。综纤维素和硫酸木质素含量测定结果显示,转基因烟草纤维素含量增加,木质素含量减少,表明双价基因能够有效表达。     

反义4CL与UGPase双价基因在烟草中的转化及表达分析

构建了携带紫穗槐尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase, UGPase)基因和4-香豆酸: 辅酶A连接酶(4-coumarate: coenzyme A ligase, 4CL)反义基因的双价基因植物表达载体, 用热激法转化至根癌农杆菌LBA4404中, 并用制备的农杆菌工程菌进行了烟草转化。PCR及Southern 杂交结果证实, 双价基因已成功整合到烟草基因组中。综纤维素和硫酸木质素含量测定结果显示, 转基因烟草纤维素含量增加, 木质素含量减少,表明双价基因能够有效表达。     

Developmental Control of Lignification in Stems of Lowland Switchgrass Variety Alamo and the Effects on Saccharification Efficiency

The switchgrass variety Alamo has been chosen for genome sequencing, genetic breeding, and genetic engineering by the US Department of Energy Joint Genome Institute (JGI) and the US Department of Energy BioEnergy Science Center. Lignin has been considered as a major obstacle for cellulosic biofuel production from switchgrass biomass. The purpose of this study was to provide baseline information on cell wall development in different parts of developing internodes of tillers of switchgrass cultivar Alamo and evaluate the effect of cell wall properties on biomass saccharification. Cell wall structure, soluble and wall-bound phenolics, and lignin content were analyzed from the top, middle, and bottom parts of internodes at different developmental stages using ultraviolet autofluorescence microscopy, histological staining methods, and high-performance liquid chromatography (HPLC). The examination of different parts of the developing internodes revealed differences in the stem structure during development, in the levels of free and well-bound phenolic compounds and lignin content, and in lignin pathway-related gene expression, indicating that the monolignol biosynthetic pathway in switchgrass is under complex spatial and temporal control. Our data clearly show that there was a strong negative correlation between overall lignin content and biomass saccharification efficiency. The ester-linked p-CA/FA ratio showed a positive correlation with lignin content and a negative correlation with sugar release. Our data provide baseline information to facilitate genetic modification of switchgrass recalcitrance traits for biofuel production.     

Pix Plus and Mepiquat Chloride Effects on Physiology, Growth, and Yield of Field-Grown Cotton

   apd: 2 May 2001     

Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5'-DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.     

茶树4CL基因家族的全基因组鉴定及表达分析

4-香豆酸辅酶A连接酶(4CL)是茶树苯丙烷代谢途径的核心酶,是类黄酮和木质素生物合成的关键酶,对茶树抗胁迫及茶叶品质形成有重要作用。研究通过生物信息学分析,从‘黄棪’和‘铁观音’茶树中分别鉴定出8个和9个4CL基因家族成员,命名为HD-Cs4CL1～8和TGY-Cs4CL1～9。系统发育分析将其分为A和B两组,A组中第Ⅰ亚家族的HD-Cs4CL3、HD-Cs4CL7、TGY-Cs4CL2和第Ⅱ亚家族的HD-Cs4CL2、TGY-Cs4CL5可能分别参与木质素和类黄酮合成。启动子顺式元件分析表明4CL基因中存在大量与植物发育、激素和胁迫响应相关的顺式作用元件。不同组织转录组数据分析表明,Cs4CL基因在茶树特定发育时期具有重要作用。实时荧光定量检测表明,HD-Cs4CL6和HD-Cs4CL8在GA、MeJA、ABA、低温和干旱处理下表达量均显著上调;HD-Cs4CL7在ABA处理和干旱处理下表达量上调,HD-Cs4CL2和HD-Cs4CL3在ABA处理下表达量上调。研究为进一步探究茶树4CL基因家族参与响应多种环境的生物学机制提供参考。     

甘薯4-香豆酸辅酶A连接酶基因的生物信息学鉴定和表达分析

4-香豆酸辅酶A连接酶(4-coumarate:CoA ligase, 4CL)在苯丙烷类代谢途径中具有重要调控作用。该研究利用转录组数据在甘薯品种QS48中鉴定了11个4CL基因,并将这些基因命名为Ib4CL1～Ib4CL11。序列比对和功能域分析发现, 11个Ib4CL蛋白均具有典型的4CL结构特征,包含保守的BoxⅠ和BoxⅡ的结构域。进化和保守基序分析显示,Ib4CL1和Ib4CL2属于Ⅰ类4CL,Ib4CL3属于Ⅱ类4CL,而Ib4CL4～Ib4CL11属于4CL类似蛋白。转录组数据和荧光定量PCR分析显示,Ib4CL3和Ib4CL10在甘薯QS48的叶中转录表达水平显著高于茎和块根,与绿原酸的积累呈正相关关系;Ib4CL4和Ib4CL8在嫩叶中表达较高,与花青素的积累密切相关。研究结果为进一步揭示甘薯4CL基因在苯丙烷类代谢途径中的功能奠定了基础。     

Expression of the Bovine Growth Hormone Alters the Root Morphology in Transgenic Tobacco Plants

The bovine growth hormone (bGH) is a natural peptide hormone that controls the differentiation, growth and metabolism, and is produced in the pituitary gland of cows. For the production of bGH from plants, two different bgh clones, of which the pGAbGH1 contaions only mature peptide sequences and the pGAbGH15 contains signal sequences and the first intron, as well as mature peptide sequences, were used. Those bghs under the control of the CaMV 35S promoter and NOS terminator were introduced to tobacco plants via Agrobacterium tumefaciens-mediated transformation. By PCR analyses using bgh and nptII specific primers, 17 and 21 putative transformants were respectively selected from pGAbGH1- and pGAbGH15-transformed tobacco plants. Northern blot analysis showed that the most of the transgenic lines expressed the bgh mRNA. Western blot analysis revealed that the pGAbGH1-transformed tobaccos produced recombinant bGH, but pGAbGH15-transformed ones did not produce the protein. Interestingly, some morphological changes were observed in the roots of transgenic tobacco plants. The transgenic tobacco plants had thick and short roots containing few root hairs in contrast to the non-transformed wild type plants.