Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.     

沉默马铃薯Sgt1-3基因减少块茎糖苷生物碱积累

马铃薯茄啶 糖基转移酶（solanidine glycosyltransferase,Sgt）家族成员Sgt1、Sgt2和Sgt3参与糖苷生物碱（glycoalkaloids,GAs）合成。已有研究证明,抑制该家族任一成员表达可影响马铃薯块茎中糖苷生物碱合成;然而,对Sgt家族成员的单基因实施调控很难有效降低块茎中总糖苷生物碱的积累。为降低马铃薯块茎中总糖苷生物碱的含量,本研究拟采用RNAi技术,对糖苷生物碱合成代谢途径末端酶基因家族成员Sgt1 3在转录水平进行共调控。为实现这一目的,构建了块茎特异性启动子Patatin驱动的,以Sgt1、Sgt2和Sgt3基因为靶向的RNAi表达载体pCEI-PFR,采用农杆菌介导法转化马铃薯茎段,获得10株可沉默Sgt1、Sgt2和Sgt3基因表达的Patatin RNAi融合基因的转基因植株。实时定量PCR（RT-qPCR）结果显示,Sgts基因的相对表达量分别降低了大约32%～60%（Sgt1）、29%～55%（Sgt2）和25%～66%（Sgt3）,而草甘膦抗性基因--5-烯醇式丙酮酸-3-磷酸莽草酸合酶（5-enolpyruvylshikimate-3-phosphate synthase, EPSPS）的表达量则增加了约48%～135%。高效液相色谱法（HPLC）证明,尽管转基因株系的绿色组织中糖苷生物碱含量与野生型无显著差异,但块茎中糖苷生物碱分别比野生型降低了46%～59%（庄薯3号）和42%～62%（Favorita）。上述结果提示,复合沉默茄啶 糖基转移酶家族基因可降低马铃薯块茎中糖苷生物碱的积累。此外,该结果可能对研究马铃薯不同组织间糖苷生物碱的分布和积累,以及马铃薯种质资源的创新开发具有一定启示。     

Potassium channels in the mitochondria of unicellular eukaryotes and plants

The functional characterisation of potassium channels found in the mitochondria of plants and unicellular eukaryotes is critically discussed herein, with a focus on the ATP-sensitive potassium channel and the large-conductance Ca²⁺-activated potassium channel (mitoBK_Ca channel). The physiological functions of these channels are not completely understood. We discuss the functional connections and roles of potassium channels, uncoupling protein and alternative oxidase, three energy-dissipating systems that exist in the mitochondrial respiratory chain of plants and some unicellular eukaryotes, which include preventing the production of reactive oxygen species.     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。     

Developmental regulation of beta-1,3-galactosyltransferase-1 gene expression in mouse brain

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non-negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H₂O₂ and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool.     

高等植物赤霉素2-氧化酶基因的克隆、表达及其调控

赤霉素(GA)是一类重要的植物激素,对高等植物整个生命周期的生长发育起关键作用。调控赤霉素生物合成和代谢途径中的关键酶基因的表达可以控制植物体内赤霉素的含量。GA2-氧化酶是调节赤霉素合成和代谢的关键酶之一,使活性GA失活。本文主要对GA2-氧化酶基因的克隆、表达调控及其在植物基因工程中的应用等方面进行综述,为通过基因工程技术调控植物体内活性赤霉素的含量从而得到改良品种提供思路。     

Firefly luciferase as a reporter of regulated gene expression in higher plants

The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue makes it a particularly useful marker in transient or stable transformation experiments.     

Analysis of RNA Silencing in Agroinfiltrated Leaves of <Emphasis Type="Italic">Nicotiana Benthamiana</Emphasis> and <Emphasis Type="Italic">Nicotiana Tabacum</Emphasis>

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.     

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS gene dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially down mutations in essential genes where only a short 5 sequence of the mRNA is required. They also suggest that the position effect on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

The light-dependent regulation of gene expression during plastid development in higher plants

In recent years there has been a considerable increase in our understanding of the manner by which light affects gene expression during chloroplast development. In most systems that have been studied, light acts through sensitive photoreceptor molecules and quantitatively increases or represses the level of expression of specific nuclear-and plastid-encoded genes. Although the mechanisms are obscure, a picture is beginning to emerge in which the coordination of nuclear and plastid gene expression is controlled by regulatory mechanisms originating within their respective subcellular compartments. This review summarizes some of our current knowledge concerning the nature of light-regulated gene expression in higher plants and provides a prospectus for future research in this area.     

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.