Substrate Infiltration and Histological Fixatives Affect the In Situ Localisation of β‐Glucuronidase Activity in Transgenic Tissues

The β‐glucuronidase (GUS) gene is a widely used reporter gene in transgenic research. This study shows that although histochemical localisation of GUS activity may be very specific, differences in incubation conditions and tissue status can lead to artificial localisations that are independent of gene activity. The objective of the current studies was to evaluate the factors that affect the in‐situ localisation of β‐glucuronidase using transgenic tobacco plants as model tissues. The aspects considered include tissue size as well as and the addition of surfactants, vacuum infiltration and chemical fixatives. Transgenic tobacco plants exhibited variable staining patterns dependent on the size of tissue assayed and the treatments that affected the infiltration of substrate. A gradient of blue staining was observed in larger tissue pieces (10 mm²), where staining in central areas was light blue in contrast to edges, which stained deep indigo. More intense staining was associated with peripheral cell layers and regions adjacent to leaf veins. Thinner tissue strips incubated under similar conditions exhibited intense and even X‐Gluc staining. Addition of Triton X‐100 (1%) surfactant and vacuum infiltration (2 min) produced considerably quicker and more uniform staining (intense and consistent indigo blue colour) of the examined tissue after a 4 to 6‐h incubation. Chemical fixation of tissues before GUS assay resulted in quantitative and histochemical differences in enzyme activity that were dependent on the fixative type and duration. Quantitative measurements using the MUG fluorometric assay showed that Histochoice? provided the highest retention of GUS activity, maintaining more than 80 and 50% of the activity after fixation for 15 and 30 min, respectively. Activity in decreasing order was obtained with paraformaldehyde, glutaraldehyde, ethanol and FAA. GUS activity was affected not only by the type of fixative, but also by the duration of fixation with longer fixation producing lower GUS activity. From the experiments performed it can be concluded that those treatments that enhance substrate penetration, i.e., the addition of surfactant and vacuum infiltration, improve the consistency and speed of X‐Gluc staining.     

APICAL FOLIAR NECTARY OF POMEGRANATE (PUNICA GRANATUM: PUNICACEAE)

This is the first report of an extrafloral nectary in the Punicaceae. Each leaf of pomegranate bears a single apical nectary that consists of a closely packed spherical mass of densely staining cells, and of a surrounding layer of cells that are larger and more vacuolate, though still densely staining. The epidermis at the apex of the nectary (which is also comprised of small, densely staining cells) bulges to form a bump over a small chamber. The leaf apex lacks stomata or other specialized pores for nectar to escape. However, separation of epidermal cells and conspicuous ruptures sometimes occur. The midvein and 2–4 lateral bundles converge on the nectary. The xylem stops before reaching the nectary, but the phloem continues, curving about halfway around the nectariferous tissue. Chromatographic analysis of nectar droplets showed roughly equal amounts of fructose, glucose, and sucrose. This nectary resembles leaf apical glands of Lafoensia in the closely related family Lythraceae.     

The expression of a baculovirus-derived chitinase gene increased resistance of tobacco cultivars to brown spot (Alternaria alternata)

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase gene coding region was amplified using the polymerase chain reaction, inserted into a plasmid (pROK‐2) and replicated in Escherichia coli XL1–blue. The recombinant plasmid was mobilised into Agrobacterium tumefaciens LBA 4404 and inoculated into tobacco leaf discs. The presence of the expressed chitinase in foliar tissue of kanamycin‐resistant plantlets of three Nicotiana tabacum cultivars (CF80, K326 and Xanthi‐nc) was inferred using immunoblotting, and enzyme activity was confirmed using a fluorometric assay. Confocal laser scanning microscopy with immunofluorescent staining of foliar sections from N. tabacum Xanthi‐nc expressing the viral chitinase indicated that the enzyme was restricted to the vascular tissue. Heliothis virescens larvae fed on leaf tissue expressing chitinase were not impaired either in their development to pupation or in their feeding behaviour, in comparision with their counterparts that had consumed similar amounts of untransformed tobacco leaf tissue. By contrast, when tobacco leaves were mechanically inoculated with Alternaria alternata, very few brown spots were observed at inoculation sites in chitinase‐expressing tissue, whereas large and spreading lesions formed in untransformed tobacco tissue. Of all lines that were transformed, as determined by kanamycin resistance, 59% had fewer symptoms of disease (smaller disease indices) than those for untransformed controls.     

Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for β-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.     

Isolation of functional extensor and flexor protoplasts fromPhaseolus coccineus L. pulvini: potassium induced swelling

Methods are described for the isolation of functional protoplasts from secondary pulvinus tissue (flexor and extensor) and from leaf mesophyll tissue of primary leaves ofPhaseolus coccineus L. Integrity of the protoplasts was shown by vital staining and their ability to evolve oxygen in the light. Extensor-cell protoplasts increased their volume for up to 60% upon addition of 10 mM KCl. This K⁺-induced swelling was accompanied by increased rates of proton extrusion.     

A direction‐selective local‐thresholding method,DSLT, in combination with a dye‐based method for automated three‐dimensional segmentation of cells and airspaces in developing leaves

Quantifying the anatomical data acquired from three‐dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi‐photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone‐oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time‐lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal‐to‐noise ratio at greater tissue depths, we also developed a local thresholding method (direction‐selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single‐cell resolution, and measured the airspace volumes in intact leaves.     

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.     

Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 μM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta? tolerance) and the reporter gene uidA coding for β-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1^–1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T₀ plants and by PCR analyses of somatic embryos produced from T₀ plants. Received: 9 April 1997 / Revision received: 11 May 1997 / Accepted: 3 June 1997     

Leaf mechanics and herbivory defence: How tough tissue along the leaf body deters growing insect herbivores

Research on herbivory defence often focuses on leaf chemistry but less on how plant mechanical properties like leaf veins deter herbivores. Herbivores often eat tough, complex plant tissue, yet how mechanical properties affect feeding performance as the consumer grows is unclear. We measured the toughness and strength of five types of leaf tissue – the midrib, the secondary and marginal veins and the lamina inside (inner) and outside (outer) the marginal vein – in mature Eucalyptus viminalis and Eucalyptus ovata leaves with punch tests. Leaf veins were, on average, 6.2 times tougher than lamina. Marginal veins were uniformly strong and tough along the leaf body, while midribs were less strong and secondary veins less tough toward leaf tips. We correlated the force required to puncture leaf tissue with the feeding performance of a chewing insect herbivore (the spiny leaf insect, Extatosoma tiaratum (Phasmida)) across four instar stages to explore the role of tough leaf veins as potential feeding barriers. Larvae more often ate less tough leaf tips and tougher tissue as they grew. However, younger larvae were capable of penetrating the tough marginal vein when starved. We suggest tough leaf veins and consumer position along the leaf body influence insect herbivore feeding performance over their lifetime.     

中亚热带樟科3种植物幼苗叶结构型性状的种间差异及其相关性

为了揭示植物叶功能性状对全球气候变化的响应和适应规律,以樟科3种植物幼苗为研究对象,将其人工采集的种子经低温沙藏并于温室培育,然后将幼苗移栽到野外同质园中自然生长,研究中亚热带樟科植物刨花楠(Machilus pauhoi)、香樟(Cinnamomum camphora)和闽楠(Phoebe bournei)两年生幼苗生长季节9种叶结构型性状的种间差异及其相关性。结果显示,(1)在3种植物幼苗中,香樟叶面积、叶厚度、叶体积、叶氮磷比最大,叶氮浓度最高,叶组织密度最小;闽楠叶组织密度、叶干物质含量最大,叶磷浓度最高,叶面积、叶体积、比叶面积、叶氮磷比最小,叶氮浓度最低;刨花楠比叶面积最大,叶厚度、叶干物质含量最小,叶磷浓度最低;3种植物幼苗的叶厚度之间均无显著差异。(2)Pearson相关分析和线性回归分析结果表明,樟科3种植物幼苗的叶面积与叶体积间呈极显著正相关(P0.01)、与叶干物质含量间呈极显著负相关(P0.01)、与叶磷浓度间呈极显著负相关(P0.01)、与叶组织密度间呈显著负相关(P0.05)、与叶氮浓度间呈显著正相关(P0.05);叶厚度与比叶面积间呈显著负相关(P0.05)、与叶干物质含量间呈显著正相关(P0.05);叶体积与叶组织密度间呈极显著负相关(P0.01)、与叶氮浓度间呈极显著正相关(P0.01)、与叶磷浓度间呈显著负相关(P0.05);叶组织密度与叶磷浓度间呈显著正相关(P0.05);比叶面积与叶干物质含量间呈极显著负相关(P0.01)、与叶磷浓度间呈极显著负相关(P0.01);叶干物质含量与叶磷浓度间呈极显著正相关(P0.01)。这表明在同质园中生长的刨花楠、香樟和闽楠幼苗的9种叶结构型性状表现出一定的种间差异,不同物种同一叶结构型性状指标的大小顺序、种间差异及其显著性不同;3种植物幼苗不同叶结构型性状之间的相关性及其显著程度也不同。本研究结果为揭示樟科3种植物幼苗叶功能性状对全球气候变化的响应和适应规律提供了基础数据。     

黄土高原不同植被带内草地植物叶片解剖性状的变异规律

叶片作为植物与外界进行物质交换的桥梁,其解剖性状能够相互协调以应对外界环境对植物生长造成的不利影响,从而反映出植物对环境变化所采取的适应策略。通过对黄土高原不同植被带(森林草原带、典型草原带、荒漠草原带)草地群落中常见115种植物(包括单子叶植物,双子叶植物,木本植物和草本植物四种功能型植物)叶片进行取样,并运用石蜡制片技术和光学显微技术获得叶片解剖性状(包括表皮厚度、栅栏组织厚度、海绵组织厚度、叶肉厚度和叶片厚度),旨在研究不同植被带内草地植物叶片解剖性状的变异规律及其与群落内物种相对优势度之间的关系,为黄土高原植被恢复和生态环境改善提供理论依据。结果表明：(1)沿着干旱梯度,从森林草原带、典型草原带到荒漠草原带,除叶肉厚度外,植物各叶片解剖性状值均呈现增大趋势,表明干旱地区叶片的旱生结构特征明显。(2)不同功能型植物叶片解剖性状与环境因子的关系各异。木本植物和草本植物的栅栏组织厚度和栅海比均与降水和土壤养分呈显著负相关关系(P<0.05)。同时,木本植物的叶片厚度与水分呈显著负相关关系(P<0.05),而草本植物表皮厚度仅与土壤养分呈显著负相关关系(P<0.05)...     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

Malate Dehydrogenases of Pisum sativum: Tissue Distribution and Properties of the Particulate Forms

Mitochondria and leaf microbodies isolated from leaves of pea (Pisum sativum) by sucrose density gradient centrifugation were each shown to have a unique form (isoenzyme) of malate dehydrogenase (EC 1.1.1.37) based on chromatographic and kinetic properties. Root organelle preparations were shown to contain only a mitochondrial malate dehydrogenase with physical and kinetic properties similar to the leaf form. The absence of a detectable root microbody malate dehydrogenase similar to the leaf enzyme, which is intermediate in electrophoretic and chromatographic properties between the mitochondrial and soluble isoenzymes, was confirmed by diethylaminoethyl cellulose column chromatography and starch-gel electrophoresis of total homogenates from leaf and root tissue. These findings tend to support the role of the leaf microbody isoenzyme in a pathway unique to photosynthetic tissue.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

A quantitative and histological comparison of gus expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue

Summary β-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.     

青藏高原草地植物叶解剖特征

运用常规石蜡制片技术对我国青藏高原66种草地植物优势种的叶解剖特征进行研究,并分析了叶解剖特征与海拔、生长季降水及生长季均温之间的关系.结果表明:青藏高原草地植物叶片具有很多适应高寒环境的结构特征,如表皮层厚且表皮细胞大小差异显著,表皮毛等表皮附属物发达,异细胞丰富,通气组织普遍发达等;叶片各组成部分厚度的变异程度不同,其中海绵组织厚度变异最大,其次为上角质层、下表皮层、下角质层、上表皮层、栅栏组织,叶片厚度的变异最小;青藏高原草地植物叶片各组成部分的厚度存在协同进化,上下角质层厚度呈强烈正相关,海绵组织厚度与叶片厚度相关性最强;青藏高原草地植物叶片各组成部分的厚度与海拔、生长季降水、生长季均温3个重要环境变量呈较弱的相关性,总体表现为随海拔升高叶片各组成部分的厚度减小,而随生长季降水和生长季均温的增加叶片厚度增加.     

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.