Comparative transcriptional analysis reveals differential gene expression between Sand Daffodil tissues.

Sand Daffodil (Pancratium maritimum) is a world-wide endangered Amayllidaceae species and represents an important anti-cancer medicinal resource due to alkaloids production. Despite its increasing pharmaceutical importance, there are not molecular resources that can be utilized toward improving genetic traits. In our research, the suppression subtractive hybridization (SSH) method conducted to generate large-scale expressed sequence tags (EST), was designed to identify gene candidates related to the morphological and physiological differences between the two tissues, leaves and bulbs, since lycorine, the main anti-cancer compound, is there synthesized. We focused on identification of transcripts in different tissues from Sand Daffodil using PCR-based suppression SSH to identify genes involved in global pathway control. Sequencing of 2,000 differentially screened clones from the SSH libraries resulted in 136 unigenes. Functional annotation and gene ontology analysis of up-regulated EST libraries showed several known biosynthetic genes and novel transcripts that may be involved in signaling, cellular transport, or metabolism. Real time RT-PCR analysis of a set of 8 candidate genes further confirmed the differential gene expression.

In situ metabolic profiling of single cells by laser ablation electrospray ionization mass spectrometry.

Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.

Necessity of high temperature for the dormancy release of Narcissus tazetta var. chinensis.

Winter dormancy has been extensively studied in many plants, while much less information is available for summer dormancy. Narcissus tazetta var. chinensis is characterized by a prolonged period of summer dormancy during the annual cycle. In the present study, we characterized the nature of dormancy in the controlled growth conditions and investigated the effects of temperature and ethylene on dormancy release. Cessation of growth and senescence of aerial tissues occurred even under conditions favorable for growth, suggesting an endo-dormancy process. Bulbs failed to sprout when they were exposed to low storage temperatures, while high temperature treatment preceding low storage temperatures, or heating interruption during low storage temperatures, could make bulbs sprouting. These results suggest that high temperatures are necessary for endo-dormancy release. Ethylene application during a later storage stage showed an obvious accelerative effect on bulb sprouting, whereas ethylene application during the middle stage had no effect on sprouting. The biological progression of dormancy was further studied through cytological and physiological analyses. Under natural conditions, the ethylene level was reduced to trace amounts with the transition and progression of dormancy. A transient peak in ethylene release was found when the plugged plasmodesmata (PD) began to re-open and flower initiation began. The most common PD possessed electron-dense deposits in endo-dormancy. These data indicate that endo-dormancy ends when flower initiation begins and ethylene peak occurs. Ethylene application had no effect on dormancy release, while it hastened sprouting only after the release from endo-dormancy by high temperature.

Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis.

Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation.