High speed HPLC analysis of polyamines in plant tissues

A high speed high performance liquid chromatography (HPLC) method for the quantification of putrescine, spermidine, and spermine in biological samples is described. The dansylation is followed by a sample cleanup. The isocratic HPLC-analysis with acetonitrile:H₂O (72:28 volume/volume) on 10 centimeter long, 3 micrometer octadecyl silica columns (3 millimeter inner diameter) takes only 4.5 minutes. By our method about 100 analyses can be done in 1 day.     

Cytochemical methods for the localization of invertase activity in plant tissues

Summary Two cytochemical methods for the localization of acid and alkaline invertases are given. The first is based upon the reduction of a silver complex at two different pH ranges, whilst the second is based upon the tetrazolium raction and permits quantification of the rate of activity of alkaline invertase activity. The distribution of alkaline invertase activity throughout the root apex of Pisum sativum and the cell wall localization of acid invertase for material excised from tuber tissue of Helianthus tuberosus are both confirmed.     

Cytochemical methods for the localization of invertase activity in plant tissues

Two cytochemical methods for the localization of acid and alkaline invertases are given. The first is based upon the reduction of a silver complex at two different pH ranges, whilst the second is based upon the tetrazolium reaction and permits quantification of the rate of activity of alkaline invertase activity. The distribution of alkaline invertase activity throughout the root apex of Pisum sativum and the cell wall localization of acid invertase for material excised from tuber tissue of Helianthus tuberosus are both confirmed.     

Chemical methods of estimating plant available manganese in soils

Summary Twelve different chemical extraction procedures for extracting soil manganese were used. Soil test values determined for fourteen representative soil samples of Rajasthan State with manganese uptake by six crop species have shown that of the extractants used, 3N ammonium dihydrogen orthophosphate can be best used for estimating plant available soil manganese.     

Elemental concentrations in plant tissues as influenced by low pH soils

Summary Soils influenced by acid mine drainage (pH<5.0) are characterized by low concentrations of essential nutrients and increased solubility of heavy metals. The conditions typically reduce plant establishment and growth. However, river birch (Betula nigra L.) is commonly found along low pH streams in southeastern Ohio. The objective of this study was to determine the concentration of Al, Mn, Ca and Mg inB. nigra tissues.The results indicate Al and Mn are accumulating inB. nigra when compared to other species. Within river birch, Al concentrations are highest in roots; Mn concentrations are highest in leaves. There is not a concomitant reduction in Ca and Mg concentrations as suggested by soil levels.     

Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues

Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite.     

Histochemical methods for detection of heavy metals and strontium in the tissues of higher plants

Elaboration and application of histochemical methods for detection of heavy metals (Cd, Pb, Ni, Zn) and strontium for the purpose of investigating their distribution, accumulation, and translocation within the tissues of higher plants are discussed. Detailed protocols of metal detection with metallochrome indicators dithizone (Cd, Pb), dimethylglyoxime (Ni), sodium rhodizonate (Sr), zincon (Zn), and fluorescent indicator Zinpyr-1 (Zn) by light and fluorescence microscopy are described. Special attention is given to interpretation of the obtained results, advantages and drawbacks of these methods, as well as potential problems associated with histochemical analysis of distribution of heavy metals and strontium.     

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.     

A rapid screening technique for the detection of repeated DNA sequences in plant tissues

Summary DNA sequences cloned from nuclear and mitochondrial chromosomes have been used as hybridisation probes to distinguish different plant genotypes. The probes are hybridised to squashed segments of tissue e.g. root tips. The squash-dot method is rapid and suitable for screening large numbers of individual plants. One probe, specific for a rye repeated sequence family, enables rye chromosomes to be detected in wheat plants. A probe for ribosomal DNA enables plants with high or low numbers of ribosomal RNA genes to be distinguished. A maize mitochondrial DNA probe is used to distinguish plants with N, T or S cytoplasms.     

In situ detection of salicylic acid binding sites in plant tissues

The determination of hormone‐binding sites in plants is essential in understanding the mechanisms behind hormone function. Salicylic acid (SA) is an important plant hormone that regulates responses to biotic and abiotic stresses. In order to label SA‐binding sites in plant tissues, a quantum dots (QDs) probe functionalized with a SA moiety was successfully synthesized by coupling CdSe QDs capped with 3‐mercaptopropionic acid (MPA) to 4‐amino‐2‐hydroxybenzoic acid (PAS), using 1‐ethyl‐3‐(3‐dimethyllaminopropyl) carbodiimide (EDC) as the coupling agent. The probe was then characterized by dynamic light scattering and transmission electron microscopy, as well as UV/vis and fluorescence spectrophotometry. The results confirmed the successful conjugation of PAS to CdSe QDs and revealed that the conjugates maintained the properties of the original QDs, with small core diameters and adequate dispersal in solution. The PAS–CdSe QDs were used to detect SA‐binding sites in mung bean and Arabidopsis thaliana seedlings in vitro and in vivo. The PAS–CdSe QDs were effectively transported into plant tissues and specifically bound to SA receptors in vivo. In addition, the effects of the PAS–CdSe QDs on cytosolic Ca²⁺ levels in the tips of A. thaliana seedlings were investigated. Both SA and PAS–CdSe QDs had similar effects on the trend in cytosolic‐free Ca²⁺ concentrations, suggesting that the PAS–CdSe QDs maintained the bioactivity of SA. To summarize, PAS–CdSe QDs have high potential as a fluorescent probe for the in vitro/in vivo labeling and imaging of SA receptors in plants. Copyright © 2014 John Wiley & Sons, Ltd.     

Construction and characterization of a cloned probe for the detection of Phoma tracheiphila in plant tissues

Summary The construction and characterization of the recombinant clone, pPhoB25 is described. This clone contains an highly repeated sequence of DNA from the fungus Phoma tracheiphila, a pathogen of the lemon. The specificity of the probe was determined by molecular hybridization with the DNA of the host plant and with those of several fungi which can be found associated with the lemon plants. The clone was used successfully as a specific and sensitive probe for the detection of the pathogen in infected plant tissues by dotblot hybridization under laboratory and field conditions.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

Multiple gene detection by in situ RT-PCR in isolated plant cells and tissues

With the number of functional genomic approaches in plant biology increasing daily, the demand for rapid and reliable RNA localization techniques for gene characterization is being felt. We present herein a novel, liquid phase in situ RT-PCR (IS-RT-PCR) protocol using a combination of gene-specific fluorescent primers and spectral confocal microscopy to localize target RNA in epicotyl sections and xylogenic suspension cultures of Zinnia elegans. Potential sources of artefacts from fixation to gene detection were systematically eliminated using both fluorescent primers and nucleotides for 18S rRNA gene detection, resulting in a set of optimal parameters for IS-RT-PCR that may be readily adapted to any target gene. By judiciously choosing fluorescent primers with non-overlapping fluorochromes, we have shown that our technique is readily adapted to multiplex IS-RT-PCR, enabling the simultaneous localization of more than one gene within a complex tissue or heterogeneous cell population. A 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-HEX)-labelled primer and a tetrachloro-6-carboxy-fluorescein (TET)-labelled primer were designed for two marker genes associated with programmed cell death in tracheary elements (TEs): an endonuclease (Zen1) and a cysteine protease (ZcP4), respectively. An additional Cyan5 (Cy5)-labelled primer was used to monitor 18SrRNA expression. As expected, the 18S signal was constitutively expressed throughout epicotyls sections and living cells in xylogenic in vitro cultures, whereas Zen1 and ZcP4 were co-localized in forming TEs both in planta and in vitro. Analogous to clustering analysis of gene expression using microarrays to elucidate common metabolic pathways and developmental processes, this novel technique is perfectly adapted to gaining a better understanding of gene function via the coordinated expression of genes in specific cell types of complex tissues and cell populations.     

Two rapid fluorescence procedures for the detection of some thio pungent compounds in plant tissues

Summary Two rapid flourescence procedures are described for detecting sulphydryl, disulphide and isothiocyanate groups of scented and pungent principles present in the vacuolar sap of onion, garlic and cabbage. To localize compounds containing sulphydryl groups, fresh or fixed frozen sections of the plants were treated with mercurochrome. After the fluorochromization, strongly-positive sulphydryl sites emitted an intense orange-red fluorescence, while weakly-positive sites emitted a distinctive red-brown fluorescence. Disulphide groups were detected by first reducing with thioglycolic acid to thiol groups before treating with mercurochrome. To effect isothiocyanate localization, frozen sections were exposed to ammonia: isothiocyanates were converted to thioureas and the engendered amino groups were revealed with fluorescamine.