Plant Molecular Biology Reporter

Formosan lily (Lilium formosanum), a species endemic in Taiwan, is characterized by showy and fragrant flowers. To understand the gene expression at its reproductive phase, we constructed a suppression subtractive cDNA library of immature flower buds, from which 1,324 expressed sequence tags (ESTs) were randomly selected and sequenced. These EST sequences were clustered into 974 nonredundant sequences. Based on BLAST searching, functions of 376 sequences (39%) were determined, and 80 sequences showed high similarities to genes encoding hypothetical proteins without known functions. Another 518 sequences did not show significant homology to any known sequences and were therefore classified as novel sequences. Further analyses of the 376 ESTs sequences revealed high abundance of stress-related and flower-development genes. The highly expressing stress-related transcripts include 39 with high similarities to lipid transfer proteins, five to ascorbate peroxidases, and five to heat shock proteins 70. Using real-time quantitative RT-PCR analysis, we further revealed the expression of these three genes in the immature flower buds and in the pistils or stamens of the blooming flower of Formosan lily collected from alpine regions. These results suggest that the flower of L. formosanum possesses a significantly elevated level of stress genes in response to alpine environment and the ESTs analyzed here represent a valuable resource for studying a resistance mechanism of the reproductive organs of Formosan lily.

The original version of this article unfortunately contained missing information at author’s affiliations. The affiliation address of the author’s Nishtha Mishra and Shipra Pandey should include “Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, U.P, India” as listed below.

Presented here is a simple method that enables amplification of the DNA immediately surrounding the junction between an inserted tDNA and the host genome, without prior sequence information.

The universal stress proteins (USPs) comprise a large family of proteins that has been found and characterized in a variety of organisms including plants. Intrinsic domains of these proteins are considered as one of the most ancient domains in all life forms. The first member of the USP family was isolated and extensively characterized in bacteria during the 1990s, demonstrating that the USP-containing proteins are phosphoproteins responsive to a variety of abiotic stress stimuli. Since then, many other members of the family have been characterized. Here, we present the isolation and characterization of two USP genes from Gossypium hirsutum, the most widely cultivated cotton species that has a serious impact in world’s economy. The two cotton USP genes, each in the form of two alleles, possess the same structural organization with their Arabidopsis homologues, possessing one intron sequence separating the coding sequence into two unequal parts, a large 5′-end part and a small 3′-end tail. In silico amino acid sequence analysis and three-dimensional modeling show the high levels of conservation in the USP domain, an indication that the cotton GhUSPs may function in a variety of developmental and stress-induced pathways. Moreover, expression analysis showed that while both are expressed in stems and meristems, although at different levels, only one of them is expressed in flowers. Upon salt treatment, both GhUSPs are transcriptionally activated, with the GhUSP1 reaching a peak within 30 min after the application of the stress stimuli, whereas activation of the GhUSP2 lasts longer.

Difficulties extracting high-quality RNA from recalcitrant plant tissues are often due to high levels of phenolics, carbohydrates, or other compounds that bind and/or co-precipitate with RNA. We describe here a method using soluble polyvinylpyrrolidone (PVP) and ethanol precipitation, which has been successful in several recalcitrant systems where other specialized RNA extraction methods failed to deliver suitable product. Using this method, RNA capable of reverse-transcription/PCR amplification and cDNA library construction was isolated from ripening grape berries, dry seeds of Albizia procera and radish, and leaf tissue of A. procera and Griffonia simplicifolia. This method is applicable to a variety of plant tissues.

We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 μg DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles.

Marlene de la Cruz, Fabiola Ramirez and Hector Hernandez (1997)DNA Isolation and Amplification from Cacti. Plant Mol Biol Rep 15:319-325. p. 320: The CTAB extraction buffer should contain 500 mg ascorbic acid and 500 mg DIECA in 100 mL (final concentration of 0.5%).

The dehydration-responsive element binding (DREB) transcription factor binds to the dehydration-responsive element (DRE), a cis-acting element, and regulates the expression of multiple target genes involved in plant tolerance to drought, salinity, and cold stress. Phyllostachys edulis is one of the most widely cultivated bamboos species, which is adapted for cultivation in warm and wet condition, and is sensitive to cold. However, a DREB homolog gene has not yet been identified in P. edulis. In this paper, we report the characterization of the peDREB1, cloned from cold-induced P. edulis by rapid amplification of cDNA ends (RACE). The characterization of the gene product as a DREB transcription factor is supported the nuclear localization of a transiently expressed green fluorescent protein (GFP) fusion in onion epidermal cells. Furthermore, peDREB1 can activate reporter gene expression, and we show that the protein specifically binds to the conserved DRE element in a yeast one-hybrid assay. Expression analysis shows that peDREB1 transcription levels rapidly accumulate following exposure to cold stress, peaking at 3 h, while only small changes in mRNA expression are observed during abscisic acid, osmotic stress, or salt treatment. Thus, we have identified and characterized the peDREB1 transcription factor. Similar to other APETALA2/ethylene-responsive element-binding proteins (AP2/EREBP) regulators, we report that the peDREB1 homolog is activated in response to environmental factors in tropical plants.