a-colorimetric-method-for-measuring-iron-content-in-plants

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Ben-Gurion University of the Negev

Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.

The water- and salt-stress-regulated Asr1 (abscisic acid stress ripening) gene encodes a zinc-dependent DNA-binding protein.

Novel cationic amphiphilic compounds were prepared from vernonia oil, a natural epoxidized triglyceride, and studied with respect to vesicle formation, encapsulation of biomaterials such as DNA, and their physical stability and transport through isolated plant cuticle membranes. The amphiphiles studied were a single-headed compound III (a quaternary ammonium head group with two alkyl chains) and a triple-headed compound IV, which is essentially three molecules of compound III bound together through a glycerol moiety. Vesicles of the two amphiphiles, prepared by sonication in water and solutions of uranyl acetate or the herbicide 2,4-D (2,4-dichloropenoxy acetic acid), were examined by TEM, SEM, AFM, and confocal laser systems and had a spherical shape which encapsulated the solutes with diameters between 40 and 110 nm. Vesicles from amphiphile IV could be made large enough to encapsulate a condensed 5.2kb DNA plasmid (pJD328). Vesicles of amphiphile IV were also shown to pass intact across isolated plant cuticle membranes and the rate of delivery of encapsulated radio-labeled 2,4-D through isolated plant cuticle membranes obtained with these vesicles was clearly greater in comparison to liposomes prepared from dipalmitopyl phosphatidylcholine (DPPC) and the control, nonencapsulated 2,4-D. Vesicles from amphiphiles III and IV were found to be more stable than those of liposomes from DPPC. The data indicate the potential of vesicles prepared from the novel amphiphile IV to be a relatively efficient nano-scale delivery system to transport DNA and other bioactive agents through plant biological barriers. This scientific approach may open the way for further development of efficient in vivo plant transformation systems.

Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes.

The antiviral activity of Callissia fragrans and Simnondsia chinensis aquatic and ethanol leaf extracts, as well as purified fractions from these extracts was studied against herpetic viruses in vitro. Ethanol extract of C. fragrans effectively inhibited the infection of Vero cells by HSV-1, HSV-2 in vitro, while its aquatic extract inhibited only VZV. Although S. chinensis leaf extract strongly inhibited all studied viruses, the selectivity index of this extract was very low, due to its high toxicity. However, the majority of its fractions showed low toxicity and higher antiviral activity and therefore very high SI. Strong interactions between virus and extracts were found.

Anti-Herpetic Activity of Callissia fragrans and Simmondsia chinensis Leaf Extracts In Vitro.

Crude ethanol extracts from Ficus benjamina leaves strongly inhibit Herpes Simplex Virus 1 and 2 (HSV-1/2) as well as Varicella Zoster Virus (VZV) cell infection in vitro. Bioassay-guided fractionation of the crude extract demonstrated that the most efficient inhibition of HSV-1 and HSV-2 was obtained with the flavonoid fraction. The present study was aimed to further isolate, purify and identify substances with potent antiviral activity from the flavonoid fraction of F. benjamina extracts. Flavonoids were collected from the leaf ethanol extracts through repeated purification procedure and HPLC analysis. The antiviral activity of each substance was then evaluated in cell culture. Three known flavone glycosides, (1) quercetin 3-O-rutinoside, (2) kaempferol 3-O-rutinoside and (3) kaempferol 3-O-robinobioside, showing highest antiviral efficiency were selected and their structure was determined by spectroscopic analyses including NMR and mass spectrometry (MS). These three flavones were highly effective against HSV-1 reaching a selectivity index (SI) of 266, 100 and 666 for compound 1, 2 and 3, respectively, while the SI of their aglycons, quercetin and kaempferol amounted only in 7.1 and 3.2, respectively. Kaempferol 3-O-robinobioside showed similar SI to that of acyclovir (ACV), the standard anti-HSV drug. Although highly effective against HSV-1 and HSV-2, these flavone glycosides did not show any significant activity against VZV.

Potent antiviral flavone glycosides from Ficus benjamina leaves.

In Lilium longiflorum, vernalization is both an obligatory requirement and the major factor affecting flowering time, however, little is known about the molecular regulation of this mechanism in Lilium and other flowering bulbs. Exposure of L. longiflorum bulbs to 9 weeks at 4°C greatly promoted stem elongation within the bulb, floral transition and flowering. Subtraction libraries of vernalized (V) and non-vernalized (NV) bulb meristems were constructed. 671 and 479 genes were sequenced, from which 72 and 82 proteins were inferred for the NV-V and the V-NV libraries, respectively. Much lower transcription levels and putative gene functions were recorded in the NV-V libraries compared the V-NV libraries. However, a large number of genes annotated to transposable elements (TEs), represented more than 20% of the sequenced cDNA were expressed in the NV-V libraries, as opposed to less than 2% in the V-NV libraries. The expression profile of several genes potentially involved in the vernalization pathway was assessed. Expression of LlSOC1, the lily homologue of SUPPRESSOR OF OVER-EXPRESSION OF CO1 (SOC1), an important flowering gene in several plant species, found in the V-NV library, was highly up-regulated during bulb meristem cold exposure. The subtraction libraries provided a fast tool for relevant gene isolation. 

Characterization of expressed sequence tags from Lilium longiflorum in vernalized and non-vernalized bulbs.

Vernalization is an obligatory requirement of extended exposure to low temperatures to induce flowering in certain plants. It is the most important factor affecting flowering time and quality in Easter lily (Lilium longiflorum). Exposing the bulbs to 4 °C gradually decreases flowering time up to 50% compared to non-vernalized plants. We aim to understand the molecular regulation of vernalization in Easter lily, for which we characterized the global expression in lily bulb meristems after 0, 2, 5, 7 and 9 weeks of incubation at 4 °C.

Whole transcriptome profiling of the vernalization process in Lilium longiflorum (cultivar White Heaven) bulbs.

In deciduous fruit trees, the effect of chilling on flowering has mostly been investigated in the "indirect flowering" group, characterized by a period of rest between flower bud formation and blooming. In the present study, we explored the effects of chilling and chilling deprivation on the flowering of Ziziphus jujuba, a temperate deciduous fruit tree belonging to the "direct flowering" group, in which flower bud differentiation, blooming and fruit development occur after dormancy release, during a single growing season. Dormancy release, vegetative growth and flowering time in Z. jujuba cv. Ben-Li were assessed following several treatments of chilling. Chilling treatments quantitatively decreased the timing of vegetative bud dormancy release, thereby accelerating flowering, but had no effect on the time from dormancy release to flowering. Trees grown at a constant temperature of 25°C, without chilling, broke dormancy and flowered, indicating the facultative character of chilling in this species. We measured the expression of Z. jujuba LFY and AP1 homologues (ZjLFY and ZjAP1). Chilling decreased ZjLFY expression in dormant vegetative buds but had no effect on ZjAP1expression, which reached peak expression before dormancy release and at anthesis. In conclusion, chilling is not obligatory for dormancy release of Z. jujuba cv. Ben-Li vegetative buds. However, the exposure to chilling during dormancy does accelerate vegetative bud dormancy release and flowering. 

Dormancy release and flowering time in Ziziphus jujuba Mill., a "direct flowering" fruit tree, has a facultative requirement for chilling.

Familiar fragrances usually induce positive mood states and elicit favorable evaluation. Relaxation is also widely thought to improve mood state. Yet experimental evidence on the effect of two different stimuli, fragrance smelling and breathing relaxation, on mood state, and fragrance evaluation is lacking. This study aimed to test (1) the effect of two familiar fragrances, lavender and myrtle, and two exotic fragrances, bergamot and ravensara, on perceived mood states before and after relaxation, (2) the effect of relaxation on perceived mood states for each fragrance, and (3) the effect of relaxation on fragrance evaluation as defined by adjectives. We hypothesized that mood states and assessment of the fragrances would differently be affected both in familiar vs. non-familiar fragrances and also before and after relaxation. Participants ( = 127) completed questionnaires on their mood states at baseline (T0). They were then presented with each of the four fragrances separately and asked to report on mood state and to assess the fragrances with adjectives before (T1) and after (T2) breathing relaxation. Analyses of the T0-T1 delta values of mood states by ANOVA repeated measures and  comparisons showed that mood states were affected by fragrance smelling with no clear differences observed between familiar and exotic fragrances. The same analyses of T1-T2 values showed no differences in mood state after breathing relaxation and fragrance smelling. Fragrance assessment by adjectives indicated a non-conclusive trend for familiar and exotic fragrances. In sum, mood states induced by the fragrance smelling stimulus (T0-T1) were not changed by the addition of the second stimulus of relaxation (T1-T2), indicating that the former stimulus was stronger than the latter. On the other hand, the cognitive component represented by adjective-based assessment of fragrances was slightly modified by the relaxation stimulus.

Changes in Mood States Are Induced by Smelling Familiar and Exotic Fragrances.

Floral induction in Tulipa gesneriana and Lilium longiflorum is triggered by contrasting temperature conditions, high and low temperature, respectively. In Arabidopsis, the floral integrator FLOWERING LOCUS T (FT), a member of the PEBP (phosphatidyl ethanolamine-binding protein) gene family, is a key player in flowering time control. In this study, one PEBP gene was identified and characterized in lily (LlFT) and three PEBP genes were isolated from tulip (TgFT1, TgFT2 and TgFT3). Overexpression of these genes in Arabidopsis thaliana resulted in an early flowering phenotype for LlFT and TgFT2, but a late flowering phenotype for TgFT1 and TgFT3. Overexpression of LlFT in L. longiflorum also resulted in an early flowering phenotype, confirming its proposed role as a flowering time-controlling gene. The tulip PEBP genes TgFT2 and TgFT3 have a similar expression pattern in tulip, but show opposite effects on the timing of flowering in Arabidopsis. Therefore, the difference between these two proteins was further investigated by interchanging amino acids thought to be important for the FT function. This resulted in the conversion of phenotypes in Arabidopsis upon overexpressing the substituted TgFT2 and TgFT3 genes, revealing the importance of these interchanged amino acid residues. Based on all obtained results, we hypothesize that LlFT is involved in creating meristem competence to flowering-related cues in lily, and TgFT2 is considered to act as a florigen involved in the floral induction in tulip. The function of TgFT3 remains unclear, but, based on our observations and phylogenetic analysis, we propose a bulb-specific function for this gene.

Tulipa gesneriana and Lilium longiflorum PEBP Genes and Their Putative Roles in Flowering Time Control.

Large bulbs of Lilium longiflorum have an obligatory cold requirement to flower. Bulb cooling is widely used to induce and accelerate flowering. However, in-depth investigations of the effect of bulb cooling on major landmarks of plant development are lacking. It has been demonstrated that low temperature induces carbohydrate degradation, yet integrative studies on metabolic changes occurring in the bulb are not available. We detected that cold exposure mainly hastened bulb sprouting, rather than floral transition or blooming. Metabolite profiling of cooled and non-cooled bulbs was carried out, revealing cold-induced accumulation of soluble sugars, lipids and specific amino acids, and a significant reduction in tricarboxylic acid (TCA)-cycle elements. We observed that metabolic pathways located in the cytosol - including glycolysis, lipid synthesis and part of the gamma-Aminobutyric acid (GABA) shunt - were enhanced by cold exposure, while mitochondrial metabolism - namely the TCA cycle - was reduced by cold. We suggest a physiological model accounting for this metabolic discrepancy.

The metabolic (under)groundwork of the lily bulb toward sprouting.

This paper phenomenologically and qualitatively explores the relationship between humans and flowers as a relationship that throws light on the synergetic dynamics of embodied aesthetics. Its methods include qualitative description and thematic analyses of preferred flower types, as well as concept maps of the general term 'flower' by 120 students in Israel. The results revealed the interactive perceptual-compositional elements, as well as embodied, relational, and socially embedded elements of the aesthetic pleasure associated with flowers. Implications of this case study are generalized to understand the multiple and interactive components of embodied aesthetic experiences as a deep source of pleasure through interactive stimulation by and connection to the natural world.

Humans' Relationship to Flowers as an Example of the Multiple Components of Embodied Aesthetics.

Lilium × formolongi is a facultative long day (LD) plant. Aiming to dissect the molecular regulation of the photoperiodic pathway, largely unknown in Lilium, we explored the CONSTANS/FLOWERING LOCUS T (CO/FT) module, a major regulatory factor in the external coincidence model of photoperiodic flowering pathway in lily. We identified eight CONSTANS-LIKE (COL) family members in L. × formolongi, which could be divided into three types, according to their zinc-finger (B-box) protein domains. Type I included only LfCOL5, containing two B-box motifs. Type II contained six LfCOLs members that had only one B-box motif. Type III solely contained LfCOL9 that showed a normal B-box and a second divergent B-box motif. Phylogenic analyses revealed that LfCOL5 was the closest to the Arabidopsis CO. LfCOL5, LfCOL6 and LfCOL9 were up-regulated at the flowering induction stage under LD, coinciding with an increase in LfFT1 expression. LfCOL5, LfCOL6 and LfCOL9 also showed obvious diurnal expression pattern for three days under LD. However, under short day (SD) conditions, LfCOL5, LfCOL6 and LfCOL9 expression patterns were variable and complex, with regard to the developmental stages and circadian rhythm. LfCOL5, LfCOL6 and LfCOL9 complemented the late flowering phenotype of the co mutant in Arabidopsis. Taken together, the results suggest that LfCOL5, LfCOL6 and LfCOL9 are involved in triggering flowering induction under LD. LfCOL6 and LfCOL9 belong to different types than functional COL homologs in other plant species, illustrating the variation in phylogeny, evolution and gene function among LfCOL family members.

Functional and Evolutionary Characterization of the CONSTANS-LIKE Family in Lilium × formolongi.

Adaptation of the agrobacterium-mediated floral dipping technique is limited to date to a small number of plants. Here we present efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranking from 0.2 and 2.9%, well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre-anthesis or 3-5 days post-anthesis with 1.5% and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of XP flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrate Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. Here we demonstrate floral dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel. This article is protected by copyright. All rights reserved.

Michele Zaccai

Department of Life Sciences

Ben-Gurion University of the Negev

A Colorimetric Method for Measuring Iron Content in Plants