Anikó Páy

Transmission of paternal chloroplasts in Nicotiana

SummaryTransmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia ♀xN. plumbaginifolia Np(SR1)3 ♂ and the N. plumbaginifolia Np(gos)29 ♀xN. tabacum SR1 ♂ crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.

A 268 bp upstream sequence mediates the circadian clock-regulated transcription of the wheat Cab-1 gene in transgenic plants.

We previously reported that the expression of the wheat Cab-1 gene is regulated by an endogenous circadian rhythm and by the photoreceptor phytochrome both in wheat and in transgenic tobacco plants. To define regulatory elements necessary for the circadian rhythm-regulated Cab-1 gene expression, we now analysed the fluctuation of steady-state mRNA levels in a series of 5′ deletion mutants in transgenic tobacco plants. We found that the expression of a deletion mutant containing 211 bp upstream sequence still exhibited circadian rhythm. Furthermore we show that an enhancer-like sequence of the Cab-1 promoter (from −357 to −90) can endow a chimaeric gene consisting of a truncated 35S promoter (from −90 to +8) and the bacterial β-glucuronidase (GUS) gene with circadian clock-regulated gene expression. Finally we demonstrate by nuclear run-off experiments that the transcription rates of the Cab genes in wheat oscillate in a rhythmic manner, with a periodicity of approximately 24 hours. Consistent with our previous findings these results (i) indicate that the expression of the wheat Cab-1 gene is regulated mainly at the transcription level and (ii) identify a short promoter region between −211 and −90 that is responsible for the circadian clock-regulated gene expression.

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

SummaryChloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F1 and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis

This work shows that CRK5, a plasma membrane–associated member of the Arabidopsis Ca2+/calmodulin-dependent kinase-related protein family, phosphorylates the hydrophilic loop of PIN2 and is required for proper polar localization of PIN2 in the transition zones of roots. Inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of roots and shoots. CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

Imaging Fluorescence Detected Linear Dichroism of Plant Cell Walls in Laser Scanning Confocal Microscope

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence‐detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity‐difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber‐laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture.

A rapid method for purification of organelles for DNA isolation: self-generated percoll gradients

Self-generated Percoll gradients have been used for rapid purification of crude chloroplasts and mitochondria, obtained by common differential centrifugation techniques. Such purified organelles were used for isolating DNA from safflower (Carthamus tinctorius L.), carrot (Daucus carota L.), various Solanaceae, and numerous somatic hybrids. The method is simple, has the advantage of not requiring DNase, and is particularly well suited when only limited amounts of aseptically grown shoots are available. As judged by restriction enzyme analyses and chloroplast DNA cloning experiments, the DNAs are of sufficient purity for many molecular biological applications without CsCl gradient purification.

The mitochondrial genome of safflower: Isolation and restriction fragment analysis of DNA from CMS and restorer lines

SummaryMitochondria were isolated and purified from paired lines of safflower (Carthamus tinctorius L.) restorer and cytoplasmic male sterile plants using isopycnic gradient centrifugation in isoosmotic Percoll. Agarose gel electrophoresis of restriction endonuclease digested DNAs showed characteristic polymorphism. Restriction fragments representing about 75% of the mitochondrial genome were common to both the fertile and CMS plants, but differed significantly in stoichiometric amounts. The remaining 25% could be accounted for by unique restriction fragments observed in only one or the other plant types.

CIS-Regulatory Elements for the Circadian Clock Regulated Transcription of the Wheat CAB-1 Gene

Many genes of higher plants are expressed in a highly regulated fashion. Certain genes are expressed only at specific stages of development and only in certain cell types and/or their expression is regulated by various environmental stimuli1. In addition it has also been established that numerous physiological processes of higher plants are regulated by an endogenous circadian rhythm2. The mechanism by which an environmental stimulus or an endogenous rhythm regulates gene expression is the subject of considerable interest. Photosynthesis specific genes such as genes encoding the chlorophyll a/b binding protein (Cab) are particularly attractive as model systems to study gene regulation in higher plants. Expression of various Cab genes has been analyzed in both normal and transgenic plants. These experiments revealed that the expression of the Cab genes is induced by light and that the light induction is mediated by at least two photoreceptors, i.e. by the red absorbing phytochrome and by an as yet unidentified blue absorbing photoreceptor4. Recent studies also demonstrated that Cab mRNA levels are further modulated by an endogenous rhythm5,6,7.