Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

基于联合测序分析技术挖掘红苞凤梨lncRNA信息

为了揭示lncRNA在红苞凤梨嵌合叶片形成和生长发育过程中的调控作用机制,该文以金边红苞凤梨为材料,采用Hiseq2500测序和SMRT三代全长转录组测序联合测序分析技术,分析挖掘红苞凤梨lncRNA信息。结果表明:(1)鉴定得到6 018条lncRNA,包含3 298个基因间lncRNA,870个反义lncRNA,717个内含子lncRNA和1 109个正义lncRNA,数据量较二代测序有了极大的提高。(2)结构分析表明,红苞凤梨lncRNA的总体表达丰度低于mRNA;序列长度在400~1 200 nt区间比例高于mRNA,而在1 600 nt区间,lncRNA分布的比例显著小于mRNA; lncRNA中的外显子数量总体少于mRNA,开放阅读框长度总体上也短于mRNA。(3)差异表达分析表明,在全绿、全白叶片发育过程中鉴定到1 710个差异表达lncRNA。(4)靶基因预测结果表明,5 441个lncRNA通过cis作用方式预测到靶基因,1 544个lncRNA通过trans方式预测到靶基因。(5)靶基因的功能注释和富集分析显示,差异表达lncRNA的靶基因主要作为酶蛋白参与调节叶片代谢活动和信号转导等方面,与叶片的颜色形成、光合作用和生长发育密切相关。该文鉴定出的lncRNA信息以及对其结构和功能的分析,为红苞凤梨以及凤梨科其他植物的lncRNA表观遗传调控机理研究提供了数据基础,筛选出的差异表达lncRNA在金边红苞凤梨叶片嵌合性状的形成和生长发育中具有重要的调控作用。     

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.     

Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by the arbuscular mycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The interaction between AM fungi and plant roots is of environmental and agronomic importance. Understanding the molecular changes within the host plant upon AM fungal colonisation is a pre-requisite to a greater understanding of the mechanisms underlying the interaction. Differential mRNA display was conducted on leaf tissue of tomato plants colonised and non-colonised by the AM fungus Glomus mosseae and five putative differentially regulated cDNAs were identified. All cDNAs isolated shared high sequence similarity to known plant genes. Differential screening was initially used to establish whether the cDNAs were differentially expressed. Semi-quantitative RT-PCR was used to establish gene expression patterns for all five clones within leaf and root tissue of mycorrhizal and non-mycorrhizal colonised tomato plants. Differential regulation was observed for all five cDNAs. Down-regulation within the leaf tissue of mycorrhizal plants was observed for 4 out of the 5 cDNAs with an up-regulation observed only for one. Tissue specific regulation was observed for several cDNAs, with down-regulation observed in mycorrhizal leaf tissue and up-regulation observed within mycorrhizal root tissue as compared to non-mycorrhizal tissue.     

The Promoter of a Metallothionein-Like Gene from the Tropical Tree Casuarina Glauca is Active in Both Annual Dicotyledonous and Monocotyledonous Plants

A chimeric gene consisting of the -glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.     

Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber

We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants. Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes. Of particular interest were extremely thick roots of a type not previously described. Characterization of the transferred DNA and of the expression of the corresponding genes allowed us to determine that the genes rolABC of the TL region of the Ri plasmid are sufficient to induce thin roots similar to those observed in other species, while the aux genes of the TR region are sufficient to induce thick roots. Among clones bearing the aux genes, there was a correlation between level of expression of aux2 and root phenotype.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

A soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) polyubiquitin promoter gives strong constitutive expression in transgenic soybean

The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Developmental regulation of photosynthate distribution in leaves of rice

mRNA expression patterns of genes for metabolic key enzymes sucrose phosphate synthase (SPS), phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase, ribulose 1,5-bisphosphate carboxylase/oxygenase, glutamine synthetase 1, and glutamine synthetase 2 were investigated in leaves of rice plants grown at two nitrogen (N) supplies (N_0.5, N_3.0). The relative gene expression patterns were similar in all leaves except for 9^th leaf, in which mRNA levels were generally depressed. Though increased N supply prolonged the expression period of each mRNA, it did not affect the relative expression intensity of any mRNA in a given leaf. SPS V_max, SPS limiting and PEPC activities, and carbon flow were examined. The ratio between PEPC activity and SPS V_max was higher in leaves developed at the vegetative growth stage (vegetative leaves: 5^th and 7^th leaves) than in leaves developed after the ear primordia formation stage (reproductive leaves: 9^th and flag leaves). PEPC activity and SPS V_max decreased with declining leaf N content. After using ¹⁴CO₂ the ¹⁴C photosynthate distribution in the amino acid fraction was higher in vegetative than in reproductive leaves when compared for the same leaf N status. Thus, at high PEPC/SPS activities ratio, more ¹⁴C photosynthate was distributed to the amino acid pool, whereas at higher SPS activity more ¹⁴C was channelled into the saccharide fraction. Thus, leaf ontogeny was an important factor controlling photosynthate distribution to the N- or C-pool, respectively, regardless of the leaf N status.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-γ, 100 units/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-γ and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM; an NO donor) and/or (±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 μM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-γ and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells. H. Yamaguchi and Y. Kidachi contributed equally to this work     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

3种外源诱导剂预处理对甜瓜抗病特征及其Fom 2和CHT基因表达的影响

该研究选用水杨酸(SA)、茉莉酸甲酯(MeJA)、Ca~(2+)、无菌水(对照)作为外源预处理诱导剂,以抗、感枯萎病甜瓜品种为材料,分别于诱导预处理2d后接种甜瓜枯萎菌,并于接种5、7、9d时观察发病情况,进行病情调查;在接种后1、3、5、7、9d取甜瓜叶片,分析抗病甜瓜(MR-1)和感病甜瓜(M1-15)叶片中甜瓜抗枯萎病基因(Fom-2)、几丁质酶基因(CHT)的表达变化,以探寻提高防治甜瓜枯萎病菌侵染的技术途径。结果显示:(1)外源MeJA和SA预处理接种后2品种的病情指数显著低于对照,但Ca~(2+)处理后的病情指数与对照无显著差异。(2)经外源诱导预处理接种后,MR-1和M1-15品种叶片的Fom-2和CHT基因均出现差异表达,但Ca~(2+)诱导其上调表达的效果微弱。(3)经SA、MeJA诱导预处理接种后,2品种叶片的Fom-2和CHT基因表达总体均显著高于对照;Fom-2基因的表达抗病甜瓜MR-1分别在接种后5d、7d时达到峰值,而感病甜瓜M1-15则均在接种9d时达到峰值;CHT基因的表达抗病甜瓜MR-1则均在接种后7d时达到峰值,而感病甜瓜M1-15分别在接种后7d、9d时达到峰值。(4)Ca~(2+)处理对抗、感甜瓜叶片的Fom-2和CHT基因的表达均无显著影响。(5)相关分析表明,经SA、MeJA诱导预处理接种后,甜瓜枯萎病病情指数与Fom-2和CHT基因表达量有显著的相关性;而Ca~(2+)处理效果不显著。研究表明:SA、MeJA通过诱导Fom-2、CHT基因上调表达,进而使甜瓜的抗病性提高,而Ca~(2+)处理对两基因表达和甜瓜抗病性均无显著影响。