Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitiity to auxin

The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity.     

Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.     

Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance

The early events of transient gene expression have been investigated monitoring CAT activity in tobacco protoplasts encoded by the recombinant plasmid pRT101cat. The first appearance of CAT activity was observed within 30 minutes after the outset of cultivation, and maximal values were obtained between four and 24 hours. CAT expression, at the level of RNA synthesis, could not be inhibited by cordycepin (3deoxyadenine) added one hour after protoplast plating, whereas cycloheximide, an inhibitor of protein synthesis, showed an influence during the first four hours. This indicates a rapid decay of biologically active forms of both the DNA transferred and the CAT-mRNA synthesized within the first hours. These results suggest that in the tobacco protoplast system CAT protein stability lasts up to two weeks rather than a continuous synthesis of new enzyme.Abbreviations BAP Benzylaminopurin - CaMV Cauliflower Mosaic Virus - CAT Chloramphenicolacetyltransferase - PEG Polyethylenglycol - NAA Naphtylaceticacid     

Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells

Division and growth of most types of in vitro-cultured plant cells require an external source of auxin. In such cultures, the ratio of external to internal auxin concentration is crucial for the regulation of the phases of the standard growth cycle. In this report the internal concentration of auxin in suspension-cultured cells of Nicotiana tabacum L., strain VBI-0, was manipulated either (i) by increasing 10-fold the normal concentration of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid in the external medium; or (ii) by addition 1-N-naphthylphthalamic acid (NPA; an inhibitor of auxin efflux and of auxin efflux carrier traffic). Both treatments delayed the onset of cell division for 6-7 days without loss of cell viability. In both cases, cell division activity subsequently resumed coincident with a reduction in the ability of cells to accumulate [(3)H]NAA from an external medium. Following renewed cell division, a significant proportion of the NPA-treated cells but not those grown at high auxin concentration, exhibited changes in the orientation of new cell divisions and loss of polarity. We conclude that cell division, but not cell elongation, is prevented when the internal auxin concentration rises above a critical threshold value and that the directed traffic of auxin efflux carriers to the plasma membrane may regulate the orientation of cell divisions.     

Auxin perception and polar auxin transport are not always a prerequisite for differential growth

Using time-lapse photography, we studied the response kinetics of low light intensity-induced upward leaf-movement, called hyponastic growth, in Arabidopsis thaliana. This response is one of the traits of shade avoidance and directs plant organs to more favorable light conditions. Based on mutant- and pharmacological data we demonstrated that among other factors, functional auxin perception and polar auxin transport (PAT) are required for the amplitude of hyponastic growth and for maintenance of the high leaf angle, upon low light treatment. Here, we present additional data suggesting that auxin and PAT antagonize the hyponastic growth response induced by ethylene treatment. We conclude that ethylene- and low light-induced hyponastic growth occurs at least partly via separate signaling routes, despite their strong similarities in response kinetics.Key words: hyponastic growth, petiole, Arabidopsis, ethylene, low light, auxin, polar auxin transport, differential growthUpward leaf movement (hyponastic growth) is a trait of several plant species to escape from growth-limiting conditions.^1,2 Interestingly, Arabidopsis thaliana induces a marked hyponastic growth response triggered by various environmental stimuli, including complete submergence, high temperature, canopy shade and spectral neutral low light intensities (Fig. 1).^3–6 The paper of Millenaar et al. in the New Phytologist 2009,⁷ provides a detailed analysis of low light intensity-induced hyponastic growth and components of the signal transduction are characterized using time-lapse photography. Low light intensity-induced hyponastic growth is a component of the so-called shade avoidance syndrome. Light-spectrum manipulations and mutant analyses indicated that predominantly the blue light wavelength region affects petiole movement and fast induction of hyponastic growth to low light conditions involves the photoreceptor proteins Cryptochrome 1 (Cry1), Cry2, Phytochrome-A (PhyA) and PhyB. Moreover, we show that also photosynthesis-derived signals can induce differential growth.Open in a separate windowFigure 1Typical hyponastic growth phenotype of Arabidopsis thaliana. Side view of Columbia-0 plants treated 10 h with ethylene (5 µl l⁻¹) or low light (20 µmol m⁻² s⁻¹). Plants in control light conditions were in 200 µmol m⁻² s⁻¹. Both stimuli induce a clear leaf inclination (hyponasty) relative to the horizontal by differential growth of the petioles. Plants kept in control conditions only show modest diurnal leaf movement and leaf angles gradually decline over time due to maturation of the leaves. Note that the paint droplets were applied to facilitate quantitative measurement of leaf angle kinetics in a time-lapse camera setup.⁷The hyponastic growth response to low light intensity was not affected in several ethylene-insensitive mutant lines. Moreover, low light did not affect expression of ethylene inducible marker genes nor differences in ethylene release were noted. Therefore, we concluded that low light-induced hyponastic growth is independent of ethylene signaling. This is perhaps surprising, because ethylene is the main trigger of hyponastic growth induced by complete submergence in several species. Interestingly, both ethylene and low light can induce hyponastic growth in Arabidopsis with similar kinetics.³We showed that plants mutant in auxin perception components (transport inhibitor response1 (tir1) and tir1 afb1 afb2 afb3 quadruple, containing additional mutant alleles of TIR1 homologous F-box proteins) and plants mutant in (polar) auxin transport (tir3-1, pin-formed3 (pin3) and pin7) components had a lower hyponastic growth amplitude in low light conditions.⁷ Moreover, these mutants were less able to maintain the high leaf angles after the response maximum. Both characteristics were also noted in plants pre-treated with the polar auxin transport (PAT) inhibitor 2,3,5-triiodobenzoic acid (TIBA). We therefore concluded that auxin perception and PAT are involved in the regulation of low light-induced hyponastic growth.⁷ Interestingly, we observed that TIBA pretreatment did not inhibit ethylene-induced hyponastic growth. In fact, the response upon ethylene treatment was even modestly enhanced. In agreement with this observation, we show here that the above mentioned auxin perception and PAT mutants also showed a slightly enhanced hyponastic growth response upon ethylene treatment (Fig. 2).Open in a separate windowFigure 2Auxin involvement in ethylene induced hyponasty. Effect of exposure to ethylene (5 µl l⁻¹) on the kinetics of hyponastic petiole growth (A) in Arabidopsis thaliana Columbia-0 plants treated with 50 µm TIBa (open circles) or a mock treatment (line) adapted from Supporting Information Figure S3 of Millenaar et al. (2009)⁷ and (B–F) in Arabidopsis auxin signaling and polar auxin transport mutants (closed circles), compared to the wild type response to low light (lines). Petiole angles are pair wise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions, treatments, data acquirement and analysis see.^7,13 Error bars represent standard errors; n ≥ 12. mutants were obtained from the Nottingham Arabidopsis Stock Center (accession numbers are shown between brackets) or from the authors describing the lines. tir1-1 (n3798,¹⁴), tir1-1 afb1-1 afb2-1 (in a mixed Columbia/Wassilewskija background),¹⁵ tir3-1,¹⁴ pin3-4 (n9363,¹⁶) and pin7-1 (n9365,¹⁰).Despite that auxin and PAT are required for many differential growth responses such as phototropism and gravitropism,^8,11 these data indicate that auxin perception and PAT are not obligatory for ethylene-induced hyponasty in Arabidopsis per se. In fact, one might even conclude that auxin and PAT antagonizes ethylene-induced hyponasty. These results are partly in agreement with observations on the wetland species Rumex palustris, were pretreatment with the auxin-efflux carrier 1-naphthylphthalamic acid (NPA) resulted in doubling of the lag-phase for hyponastic growth under water, but hardly affected the amplitude of the response.¹²Together, this indicates that auxin is not always a prerequisite for differential growth responses. Based on the apparent contrasting effects of auxin perception and PAT in low light- and ethylene-induced hyponastic growth, we conclude that ethylene and low light induce hyponastic growth, at least partly, via separate signaling routes.     

Transient gene expression in tobacco protoplasts: II. Comparison of the reporter gene systems for CAT,NPT II,and GUS

The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and -Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT相似文献   

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.     

Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinuclear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time‐lapse series of the early stages of regeneration, we found that nuclear position is no prerequisite for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.     

Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts.

The auxin-regulated par gene from tobacco mesophyll protoplasts was characterized in detail to deduce its possible function. An homology search of the par gene in the NBRF databases revealed that the par gene has homology to the stringent starvation protein (ssp) gene of Escherichia coli, which is induced under starved conditions and binds in an equimolar ratio to a holoenzyme of RNA polymerase. Hence, it is supposed that the par gene product could play a similar role to that of ssp. Although sequence homology of the par gene to the Gmhsp 26-A gene from soybean was observed, both genes were shown to respond differently to plant hormones and stresses. Gmhsp 26-A is induced by heat shock, 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin and abscisic acid (ABA), whereas the par gene was induced only by auxins. Furthermore, cycloheximide treatment prevents 2,4-D-mediated accumulation of Gmhsp 26-A mRNA, but not that of par mRNA. Both par and Gmhsp 26-A respond to CdCl2, but splicing of the par pre-mRNA proceeded in a normal way, whereas splicing off the Gmhsp 26-A pre-mRNA was inhibited. Hence, the par and Gmhsp 26-A genes should have a common ancestor, but have evolved in different directions. Detailed time-course experiments confirmed that the par gene was induced immediately after the addition of auxin and expressed upon the initiation of meristematic activity in tobacco mesophyll protoplasts. As the par gene was induced by the sole treatment of cycloheximide, it was proposed that the par gene belongs to a category of 'superinduction' genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Promoter and genotype dependent transient expression of a reporter gene in plant protoplasts.

For analysis of expression of three different plant promoters such as CaMV 35S, rbc S and mas, compact plasmid vectors were constructed by use of beta-glucuronidase (GUS) gene and nos termination signal. The plasmid molecules were introduced into tobacco and tomato protoplasts by using the Mg2+/PEG transformation protocol described by Negrutiu et al. The transient assays revealed maximum expression two days after DNA uptake. The comparative studies show the following order of promoters mas, CaMV 35S, rbc S as far as the activity is concerned. We also detected genotype-dependent promoter activity in the case of tomato.     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Irrepressible,truncated Auxin Response Factors: Natural roles and applications in dissecting auxin gene regulation pathways

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.     

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.