1. Correlative imaging of structural biochemistry in plant and food quality research within an interoperable data acquisition platformMarjana Regvar, Boštjan Jenčič, Martin Šala, Aleš Kladnik, Iztok Dogša, Maja Koblar, Mitja Kelemen, Primož Vavpetič, Katarina Vogel-Mikuš, Ivan Kreft, Primož Pelicon, Paula Pongrac, 2025, original scientific article Abstract: Correlative imaging is a powerful tool for revealing information on cell-type structures and their biochemistry, with the potential to inform healthier food choices and improved dietary recommendations. Determination of plant structures and their structural biochemistry advances our understanding of specific structures designed to store different biomolecules within cells and tissues. Compared to the classical biochemical separation techniques, the key advantage of sequential correlative imaging techniques is in relating spatial plant (micro)structures to their biochemistry in a nondestructive manner. Sequential imaging reported here comprises six methodologies on a single sample, a cross-section of a Tartary buckwheat (Fagopyrum tataricum) grain, namely, bright-field and autofluorescence microscopy, fluorescence microspectroscopy, MeV-secondary ion mass spectrometry, micro-particle-induced X-ray emission, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, and laser ablation-inductively coupled plasma-mass spectrometry. Results confirm that the stepwise addition of the desired information across several classes of biomolecules and several spatial scales informs the quality and safety of plant-based produce across scales. Therefore, a viable workflow is proposed, enabling sequential spatial analysis of grain and highlighting plant structures' in situ specificity. The advantages and disadvantages of the selected methodologies were critically evaluated.
Keywords: autofluorescence, correlative molecular imaging, element distribution, grain tissues
Published in DiRROS: 07.11.2025; Views: 502; Downloads: 350
 Full text (1,71 MB)
This document has many files! More... |
2. Chloroplast vesiculation and induced chloroplast vesiculation and senescence-associated gene 12 expression during tomato flower pedicel abscissionMagda Tušek-Žnidarič, Maja Zagorščak, Živa Ramšak, Katja Stare, Marko Chersicola, Maruša Pompe Novak, Aleš Kladnik, Marina Dermastia, 2025, original scientific article Abstract: Abscission is a tightly regulated process in which plants shed unnecessary, infected, damaged, or aging organs, as well as ripe fruits, through predetermined abscission zones in response to developmental, hormonal, and environmental signals. Despite its importance, the underlying mechanisms remain incompletely understood. This study highlights the deleterious effects of abscission on chloroplast ultrastructure in the cells of the tomato flower pedicel abscission zone, revealing spatiotemporal differential gene expression and key transcriptional networks involved in chloroplast vesiculation during abscission. Significant changes in chloroplast structure and vesicle formation were observed 8 and 14 h after abscission induction, coinciding with the differential expression of vesiculation-related genes, particularly with upregulation of Senescence-Associated Gene 12 (SAG12) and Chloroplast Vesiculation (CV). This suggests a possible vesicle transport of chloroplast degrading material for recycling by autophagy-independent senescence-associated vacuoles (SAVs) and CV-containing vesicles (CCVs). Ethylene signaling appears to be involved in the regulation of these processes, as treatment with a competitive inhibitor of ethylene action, 1-methylcyclopropene, delayed vesiculation, reduced the expression of SAG12, and increased expression of Curvature Thylakoid 1A (CURT1A). In addition, chloroplast vesiculation during abscission was associated with differential expression of photosynthesis-related genes, particularly those involved in light reactions, underscoring the possible functional impact of the observed structural changes. This work provides new insights into the molecular and ultrastructural mechanisms underlying abscission and offers potential new targets for agricultural or biotechnological applications.
Keywords: abscission, chloroplast vesiculation, CURT1A, CV-containing vesicle, senescence-associated vacuole, ethylene, gene expression, tomato flower pedicel
Published in DiRROS: 10.01.2025; Views: 1320; Downloads: 840
Full text (10,69 MB)
This document has many files! More... |
3. Programmed cell death occurs asymmetrically during abscission in tomatoTal Bar-Dror, Marina Dermastia, Aleš Kladnik, Magda Tušek-Žnidarič, Maruša Pompe Novak, Shimon Meir, Shaul Burd, Sonia Philosoph-Hadas, Naomi Ori, Lilian Sonego, Martin B. Dickman, Amnon Lers, 2011, original scientific article Abstract: 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.
Keywords: plant cell, cell death, abscission
Published in DiRROS: 05.08.2024; Views: 1258; Downloads: 884
Full text (2,26 MB)
This document has many files! More... |
4. 1-aminocyclopropane-1-carboxylate oxidase induction in tomato flower pedicel phloem and abscission related processes are differentially sensitive to ethyleneMarko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Tanja Mrak, Kristina Gruden, Marina Dermastia, 2017, original scientific article Abstract: Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.
Keywords: abscission, ACO, cell separation, ethylene, laser microdissection, programmed cell death, tomato, ultrastructure
Published in DiRROS: 24.07.2024; Views: 1250; Downloads: 1132
Full text (5,69 MB)
This document has many files! More... |
5. The pattern of 1-aminocyclopropane-1-carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease-LX-encoding LeLX geneMarko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Amnon Lers, Marina Dermastia, 2018, original scientific article Abstract: The abscission of tomato leaves occurs in the petiole abscission zone, and its late stage includes two spatially divided processes: cell separation and programmed cell death (PCD). Both of these processes are regulated by ethylene. The last step in ethylene biosynthesis is conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene, which is catalysed by the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO); however, the location of ACO in the leaf petiole abscission zone is not known. The tomato gene LeLX encodes ribonuclease LX, which is a marker for PCD and is induced by ethylene during abscission, but its association with ACO has not been explored.
In a tomato transgenic line 1-7 with inhibited expression of LeLX showing delayed leaf abscission, the morphology and ultrastructure of the leaf petiole abscission zone was examined. In this zone of the cv.’VF36’ and of a transgenic line 1-7, spatiotemporal differences in expression of LeACO1 and LeACO4 were analysed and ACO protein was detected immunohistochemically.
In comparison to wild-type plants, there were no obvious morphological and ultrastructural features in the abscission zone of plants of a transgenic line 1-7 before and after abscission induction. LeACO1 expression was low before abscission induction, and increased 24 h after induction, although with no apparent spatial pattern. In contrast, LeACO4 was expressed before abscission induction, and its transcript level declined 24 h after induction on the distal side of the abscission zone fracture. In the LeLX-inhibited transgenic line, there were no significant differences in LeACO1 and LeACO4 expression in the petiole abscission zone, in comparison to wild-type plants. In addition, the ACO protein was immunolocalised to the vascular tissues that traverse the petiole abscission zone in plants of wild type and of a transgenic line 1-7; and additionally in the plane of future abscission zone fracture of transgenic-line plants.
The results suggest temporal differential expression of the LeACO genes in tomato leaf petioles and vascular localisation of ACO1 protein. Additionally, the results indicate that expression of LeACO genes is not affected by suppression of the LeLX expression.
Keywords: abscission, 1-aminocyclopropane-1-carboxylate oxidase, ethylene, gene expression, localization, RNase LX
Published in DiRROS: 24.07.2024; Views: 1276; Downloads: 807
Full text (956,06 KB)
This document has many files! More... |
6. Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potatoTjaša Lukan, Maruša Pompe Novak, Špela Baebler, Magda Tušek-Žnidarič, Aleš Kladnik, Maja Križnik, Andrej Blejec, Maja Zagorščak, Katja Stare, Barbara Dušak, Anna Coll Rius, Stephan Pollmann, Karolina Morgiewicz, Jacek Hennig, Kristina Gruden, 2020, original scientific article Abstract: Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.
Keywords: immune signaling network, NADPH oxidase RBOHD, reactive oxygen species, salicylic acid, Sola-num tuberosum (potato), spatiotemporal response analysis, virus resistance, Potyvirus
Published in DiRROS: 22.07.2024; Views: 1374; Downloads: 1003
Full text (2,87 MB)
This document has many files! More... |
7. Candidate pathogenicity factor/effector proteins of ‘Candidatus Phytoplasma solani’ modulate plant carbohydrate metabolism, accelerate the ascorbate–glutathione cycle, and induce autophagosomesMarina Dermastia, Špela Tomaž, Rebeka Strah, Tjaša Lukan, Anna Coll Rius, Barbara Dušak, Timotej Čepin, Aleš Kladnik, Maja Zagorščak, Kristina Gruden, Maruša Pompe Novak, 2023, original scientific article Abstract: The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.
Keywords: autophagosome, effector, glycolysis, pathogenicity factor, StAMP
Published in DiRROS: 24.08.2023; Views: 2441; Downloads: 1186
Full text (7,84 MB)
This document has many files! More... |
8. Geographical and temporal diversity of ‘Candidatus Phytoplasma solani' in wine-growing regions in Slovenia and AustriaNataša Mehle, Sanda Kavčič, Sara Mermal, Sara Vidmar, Maruša Pompe Novak, Monika Riedle-Bauer, Günter Brader, Aleš Kladnik, Marina Dermastia, 2022, original scientific article Abstract: As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus Phytoplasma solani’ has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian–Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with ‘Ca. P. solani,’ which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.
Keywords: Bois noir, genotyping, ‘Ca. P. solani’, tuf gene, secY, survey, tuf-b2, stamp
Published in DiRROS: 10.06.2022; Views: 2654; Downloads: 1811
Full text (3,98 MB)
This document has many files! More... |
9. Rastlinske celice v urinu pri bolnikih z Brickerjevim mehurjemTanja Planinšek, Aleš Kladnik, Živa Pohar-Marinšek, Margareta Strojan Fležar, 2012, original scientific article Abstract: V citoloških vzorcih občasno opazimo različne kontaminante, kot so smukec, kristali in nitke bombažne vate. Pri rutinskem pregledovanju citoloških vzorcev urina za malignost smo občasno opazili kontaminacijo preparatov z rastlinskimi celicami. Ugotovili smo, da so bile prisotne samo pri bolnikih, ki so imeli narejen obvod urina po Brickerju. Preparate urina za rutinsko citopatološko preiskavo smo pripravili z membransko filtracijo, jih fiksirali v Delaunayu in jih pobarvali po Papanicolaouu. Pregledali smo jih s svetlobnim mikroskopom. Pripravili smo tudi preparate iz kožnih podlog podjetij Coloplast in ConvaTec, ki jih stomisti uporabljajo za pritrditev vrečke na urostomo. Samo v Coloplastovih kožnih podlogah smo našli rastlinske celice, ki so bile morfološko enake rastlinskim celicam v urinu bolnikov z Brickerjevim mehurjem. Pripravili smo tudi preparate iz različnih snovi, ki so sestavni del kožnih podlog. Rastlinske celice so bile prisotne samo v preparatih guar gumija, ki ga pridobivajo iz semen rastline Cyamopsis tetragonoloba. Rastlinske celice so se po velikosti, obliki in barvi močno razlikovale od epitelijskih črevesnih celic, ki so prisotne v urinu bolnikov z Brickerjevim mehurjem, zato bi jih tudi neizkušen presejalec težko zamenjal z displastičnimi človeškimi celicami. Pomembno pa je, da vse elemente, ki jih najdemo v celičnih vzorcih, prepoznamo in da pri neobičajnih najdbah, kot so rastlinske celice, tudi razložimo, kako je prišlo do kontaminacije vzorca.
Keywords: citološki vzorci, rastlinske celice, Brickerjev mehur, bolezni sečil
Published in DiRROS: 31.08.2018; Views: 5687; Downloads: 1384
Full text (742,42 KB) |
