Jan Jasik

SKP1–SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

Arabidopsis Snf1‐related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD‐protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1‐cullin‐F‐box) ubiquitin ligase subunit, which suppresses the skp1‐4 mitotic defect in yeast, interacts with the PRL1‐binding C‐terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1‐binding proteasomal protein, α4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co‐immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal α‐subunits show nuclear co‐localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co‐purification of epitope‐ tagged SKP1/ASK1 with SnRK, cullin and proteasomal α‐subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and α4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

SWI3 Subunits of Putative SWI/SNF Chromatin-Remodeling Complexes Play Distinct Roles during Arabidopsis Development

SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes mediate ATP-dependent alterations of DNA–histone contacts. The minimal functional core of conserved SWI/SNF complexes consists of a SWI2/SNF2 ATPase, SNF5, SWP73, and a pair of SWI3 subunits. Because of early duplication of the SWI3 gene family in plants, Arabidopsis thaliana encodes four SWI3-like proteins that show remarkable functional diversification. Whereas ATSWI3A and ATSWI3B form homodimers and heterodimers and interact with BSH/SNF5, ATSWI3C, and the flowering regulator FCA, ATSWI3D can only bind ATSWI3B in yeast two-hybrid assays. Mutations of ATSWI3A and ATSWI3B arrest embryo development at the globular stage. By a possible imprinting effect, the atswi3b mutations result in death for approximately half of both macrospores and microspores. Mutations in ATSWI3C cause semidwarf stature, inhibition of root elongation, leaf curling, aberrant stamen development, and reduced fertility. Plants carrying atswi3d mutations display severe dwarfism, alterations in the number and development of flower organs, and complete male and female sterility. These data indicate that, by possible contribution to the combinatorial assembly of different SWI/SNF complexes, the ATSWI3 proteins perform nonredundant regulatory functions that affect embryogenesis and both the vegetative and reproductive phases of plant development.

ANATOMICAL AND ULTRASTRUCTURAL EXAMINATION OF ADVENTITIOUS ROOT FORMATION IN STEM SLICES OF APPLE

Adventitious root formation in vitro in 1-mm stem slices cut from microshoots of apple cv. Jork 9 was studied using light and electron microscopy. When indole-3-butyric acid (IBA) had been added to the medium, starch grains accumulated during the first 24 h of culture in cells of the cambial region and in cells in the vicinity of vascular tissue and in the primary rays. This accumulation occurred only in the basal part of explants. After that, the nuclei in these cells were activated, and the density of the cytoplasm and the number of cell organelles increased, whereas starch was broken down. Cambium cells started to divide transversely and at 96 h, after several divisions, a continuous ring of isodiametric cytoplasmic cells had appeared around the xylem near the basal cutting surface. The cells in this ring were rich in cell structures, and did not contain large starch grains and a central vacuole. Root meristemoids regenerated from the portions of the ring that were localized in the primary rays. From the other cells in the ring, callus developed. The meristemoids did not grow into the direction of the epidermis as in shoots, but along the vascular bundles. After emergence from the cutting surface, the meristemoids were transformed into small, dome-like primordia. They developed a typical root apex with root cap, root ground meristem and tracheid connection with shoot vascular tissue.

Steedman’s Wax for F-Actin Visualization

Actin filaments are visualised by means of indirect immunofluorescence in plant tissues that were fixed in formaldehyde, embedded in low-melting polyester wax and sectioned on a microtome. The technique described here avoids usage of detergents and organic solvents and is also compatible with immunolocalization of many other antigens.

In vitro multiplication of cashew (Anacardium occidentale L.) using shoot node explants of glasshouse-raised plants

Abstract Using glasshouse-raised plants (1 month, 1 year and 5 years old), factors affecting shoot development from shoot nodes of two Brazilian and one Tanzanian elite selections of cashew (Anacardium occidentale L.) were assessed. Sprouting of buds decreased strongly with increasing age of mother plants. Solidified media, mainly when purified agar was used, gave better results than liquid medium. Murashige and Skoog salts containing 1/2-strength macroelements were the most suitable for bud sprouting and shoot elongation. Vitamins and sucrose concentration did not have a significant effect but by replacing 20 g/l sucrose with glucose or maltose all estimated parameters were improved. Gibberellins supported bud sprouting and shoot elongation but blocked rooting. Shoots developed in the presence of cytokinins were short and produced axillary branches. Activated charcoal, cultivation of explants in darkness for the first 7 days and superoptimal temperature (35 °C) decreased bud sprouting and supported shoot elongation. Microshoots rooted in vitro at a frequency of 42% when cultured for 5 days with 100 μμ indole-3-butyric acid. Over 40% of rooted microshoots survived weaning.

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

SummaryEmbryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9˚s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.

Effects of jasmonic acid and its methylester on in vitro microtuberisation of three food yam (Dioscorea) species.

Abstract The effects of jasmonic acid (JA) applied in the medium and its methylester (MeJA) applied either in the medium or as a vapour, on shoot growth and microtuber formation were evaluated in three important food yam species (Dioscorea alata, D. cayenensis, and D. rotundata). Single nodes with leaves, derived from in vitro-multiplied material, were used as explants. When delivered at higher concentrations (10 or 50 μm) both JA and MeJA suppressed node formation. Microtuberisation was supported in all three species by adding either JA or MeJA to the medium. Significant promotory effects were observed only when photoperiod, salt compositions and sucrose concentrations known to favour microtuberisation processes in yams were used. MeJA applied as a vapour strongly inhibited microtuber differentiation in D. alata on all media tested but in D. rotundata and D. cayenensis yams MeJA, also applied in the vapour phase, exhibited slight promotory effects on microtuberisation.

Somatic Embryogenesis in Pinus Nigra Arn.

The European black pine (Pinus nigra Arn.) is one of the Slovakian pines involved in somatic embryogenesis study and belongs, taxonomically, to he section Pinus of the subgenus Diploxylon (Little & Critchfield, 1969). However, in spite of a common occurrence within the subsection Sylvestris, the species was shown to be sexually incompatible with the two additional species native to Slovakia, e.g. with Pinus sylvestris and Pinus mugo (Vidakovic, 1974; Kormutak & Lanakova, 1988). As a member of the subgenus Diploxylon, P. nigra shares the features postulated by Mirov (1967) for the genetics of a whole subgenus, e.g. a high irregularity in respect to hybridization. According to Vidakovic (1974), of the 13 hybridization attempts performed so far, only 6 interspecific crosses were successful, involving the combinations with taxonomically and geographically distant species such as Pinus resinosa, Pinus heldreichii, Pinus densiflora, Pinus thunbergii, Pinus tabulaeformis, and Pinus halepensis, respectively.