Aleš Kladnik

Cytometrical Evidence That the Loss of Seed Weight in theminiature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

“If you know a thing only qualitatively, you know it no more than vaguely. If you know it quantitatively—grasping some numerical measure that distinguishes it from an infinite number of other possibilities—you are beginning to know it deeply.” (C. Sagan, Billions and Billions, 1997). Using

Evidence of Programmed Cell Death in Post-Phloem Transport Cells of the Maternal Pedicel Tissue in Developing Caryopsis of Maize

We present cellular- and ultracellular-level studies here to show developmental programmed cell death (PCD) of placento-chalazal (P-C) cell layers in maternal pedicel tissue in developing caryopses of normal seed (Mn1) and in the invertase-deficient miniature (mn1) seed mutant in maize (Zea mays). PCD was evidenced by loss of nuclei and all subcellular membranous organizations in many P-C layers. The terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) stain that is diagnostic of apoptotic-like PCD identified spatially and temporally two distinctive subdomains, which coincided with nucellar and integumental P-C layers based on their developmental origins. The early phase of PCD in the nucellar P-C was TUNEL negative and was specific to only the fertilized caryopses, indicating that the signaling for PCD in these maternal cells originated in the zygotic tissues. In fact, the initiation of PCD coincided with endosperm cellularization and was rapidly and coordinately completed prior to the beginning of the major storage phase in endosperm. Cell shape in these cell layers was also influenced by the genotype of filial endosperm. The later phase of PCD was restricted to the integumental P-C layers underneath the nucellar cells and was TUNEL positive in both genotypes. The two subdomains of the P-C layers were also distinguishable by unique cell wall-associated phenolic compounds. Based on collective evidence, we infer that the nucellar PCD may have osmolytic etiology and may lead to activation of the post-phloem transport function of the P-C layer, whereas the integumental PCD was senescent related, in particular, protecting the maturing seed against microbes that may be transported from the maternal tissue.

Human perforin employs different avenues to damage membranes

Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro.

Programmed Cell Death Occurs Asymmetrically during Abscission in Tomato

This work examines abscission in tomato and finds that it is associated with programmed cell death and expression of the RNase LX, both of which occur on the distal side of the abscission zone, indicating asymmetric processes occurring during abscission. Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.

A comparative study on the role of cytokinins in caryopsis development in the maize miniature1 seed mutant and its wild type.

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1, resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

Perforin activity at membranes leads to invaginations and vesicle formation

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.

Distribution of Potato virus Y in potato plant organs, tissues, and cells.

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.

Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions.

We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.