Ágnes Cséplo

Triazine-resistant Nicotiana mutants from photomixotrophic cell cultures

SummaryTriazine-resistant mutants have been isolated in photomixotrophic cell cultures of Nicotiana plumbaginifolia. Triazine herbicides inhibit photosynthesis and cause extensive photodestruction of chloroplasts (bleaching) in sensitive plants. Selection was based on the greening ability of the resistant cells in the presence of 10-4 M terbutryn, under normal culture conditions, but in a medium containing a low sugar concentration. In the mutant plants, as compared to wild type, two to three orders of magnitude higher concentrations of triazines resulted in inhibition of photosynthetic electron transport and greening. The resistance was inherited maternally.

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.

Selection of chloroplast mutants.

The chloroplast genome encodes a number of proteins, including thylakoid proteins and the large subunit of ribulose biphosphate carboxylase, associated with the structure and function of the chloroplast (1-2). In addition, many components of the chloroplast translational machinery, such as all of the RNAs and some of the ribosomal proteins, are coded by the chloroplast DNA. Although there have been numerous investigations into the genetics of algal chloroplasts, similar studies with higher plants have been hampered by the uniparental (maternal) pattern of transmission of chloroplasts observed in most species, and the shortage of suitable genetic markers (3,4).

Subcellular location of lincomycin resistance in Nicotiana mutants

SummaryLincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.