Harnessing the power of AI in precision medicine: NGS-based therapeutic insights for colorectal cancer cohort.

Purpose: Developing innovative precision and personalized cancer therapeutics is essential to enhance cancer survivability, particularly for prevalent cancer types such as colorectal cancer. This study aims to demonstrate various approaches for discovering new targets for precision therapies using artificial intelligence (AI) on a Polish cohort of colorectal cancer patients. Methods: We analyzed 71 patients with histopathologically confirmed advanced resectional colorectal adenocarcinoma. Whole exome sequencing was performed on tumor and peripheral blood samples, while RNA sequencing (RNAseq) was conducted on tumor samples. We employed three approaches to identify potential targets for personalized and precision therapies. First, using our in-house neoantigen calling pipeline, ARDentify, combined with an AI-based model trained on immunopeptidomics mass spectrometry data (ARDisplay), we identified neoepitopes in the cohort. Second, based on recurrent mutations found in our patient cohort, we selected corresponding cancer cell lines and utilized knock-out gene dependency scores to identify synthetic lethality genes. Third, an AI-based model trained on cancer cell line data was employed to identify cell lines with genomic profiles similar to selected patients. Copy number variants and recurrent single nucleotide variants in these cell lines, along with gene dependency data, were used to find personalized synthetic lethality pairs. Results: We identified approximately 8,700 unique neoepitopes, but none were shared by more than two patients, indicating limited potential for shared neoantigenic targets across our cohort. Additionally, we identified three synthetic lethality pairs: the well-known APC-CTNNB1 and BRAF-DUSP4 pairs, along with the recently described APC-TCF7L2 pair, which could be significant for patients with APC and BRAF variants. Furthermore, by leveraging the identification of similar cancer cell lines, we uncovered a potential gene pair, VPS4A and VPS4B, with therapeutic implications. Conclusion: Our study highlights three distinct approaches for identifying potential therapeutic targets in cancer patients. Each approach yielded valuable insights into our cohort, underscoring the relevance and utility of these methodologies in the development of precision and personalized cancer therapies. Importantly, we developed a novel AI model that aligns tumors with representative cell lines using RNAseq and methylation data. This model enables us to identify cell lines closely resembling patient tumors, facilitating accurate selection of models needed for in vitro validation.

METACASPASE8 (MC8) Is a Crucial Protein in the LSD1-Dependent Cell Death Pathway in Response to Ultraviolet Stress.

LESION-SIMULATING DISEASE1 (LSD1) is one of the well-known cell death regulatory proteins in Arabidopsis thaliana. The lsd1 mutant exhibits runaway cell death (RCD) in response to various biotic and abiotic stresses. The phenotype of the lsd1 mutant strongly depends on two other proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN-DEFICIENT 4 (PAD4) as well as on the synthesis/metabolism/signaling of salicylic acid (SA) and reactive oxygen species (ROS). However, the most interesting aspect of the lsd1 mutant is its conditional-dependent RCD phenotype, and thus, the defined role and function of LSD1 in the suppression of EDS1 and PAD4 in controlled laboratory conditions is different in comparison to a multivariable field environment. Analysis of the lsd1 mutant transcriptome in ambient laboratory and field conditions indicated that there were some candidate genes and proteins that might be involved in the regulation of the lsd1 conditional-dependent RCD phenotype. One of them is METACASPASE 8 (AT1G16420). This type II metacaspase was described as a cell death-positive regulator induced by UV-C irradiation and ROS accumulation. In the double mc8/lsd1 mutant, we discovered reversion of the lsd1 RCD phenotype in response to UV radiation applied in controlled laboratory conditions. This cell death deregulation observed in the lsd1 mutant was reverted like in double mutants of lsd1/eds1 and lsd1/pad4. To summarize, in this work, we demonstrated that MC8 is positively involved in EDS1 and PAD4 conditional-dependent regulation of cell death when LSD1 function is suppressed in Arabidopsis thaliana. Thus, we identified a new protein compound of the conditional LSD1-EDS1-PAD4 regulatory hub. We proposed a working model of MC8 involvement in the regulation of cell death and we postulated that MC8 is a crucial protein in this regulatory pathway.

MITOGEN-ACTIVATED PROTEIN KINASE 4 impacts leaf development, temperature, and stomatal movement in hybrid aspen.

Stomatal movement and density influence plant water use efficiency and thus biomass production. Studies in model plants within controlled environments suggest MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) may be crucial for stomatal regulation. We present functional analysis of MPK4 for hybrid aspen (Populus tremula × tremuloides) grown under natural field conditions for several seasons. We provide evidence of the role of MPK4 in the genetic and environmental regulation of stomatal formation, differentiation, signaling, and function; control of the photosynthetic and thermal status of leaves; and growth and acclimation responses. The long-term acclimation manifested as variations in stomatal density and distribution. Short-term acclimation responses were derived from changes in the stomatal aperture. MPK4 localized in the cytoplasm of guard cells (GCs) was a positive regulator of abscisic acid (ABA)-dependent stomatal closure and nitric oxide metabolism in the ABA-dependent pathways, while to a lesser extent, it was involved in ABA-induced hydrogen peroxide accumulation. MPK4 also affected the stomatal aperture through deregulation of microtubule patterns and cell wall structure and composition, including via pectin methyl-esterification, and extensin levels in the GC wall. Deregulation of leaf anatomy (cell compaction) and stomatal movement, together with increased light energy absorption, resulted in altered leaf temperature, photosynthesis, cell death, and biomass accumulation in mpk4 transgenic plants. Divergence between absorbed energy and assimilated energy is a bottleneck, and MPK4 can participate in the control of energy dissipation (thermal effects). Furthermore, MPK4 can participate in balancing the photosynthetic energy distribution via its effective use in growth or redirection to acclimation/defense responses.

Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress.

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

Phototropin 1 and 2 Influence Photosynthesis, UV-C Induced Photooxidative Stress Responses, and Cell Death.

Phototropins are plasma membrane-associated photoreceptors of blue light and UV-A/B radiation. The Arabidopsis thaliana genome encodes two phototropins, PHOT1 and PHOT2, that mediate phototropism, chloroplast positioning, and stomatal opening. They are well characterized in terms of photomorphogenetic processes, but so far, little was known about their involvement in photosynthesis, oxidative stress responses, and cell death. By analyzing phot1, phot2 single, and phot1phot2 double mutants, we demonstrated that both phototropins influence the photochemical and non-photochemical reactions, photosynthetic pigments composition, stomata conductance, and water-use efficiency. After oxidative stress caused by UV-C treatment, phot1 and phot2 single and double mutants showed a significantly reduced accumulation of H2O2 and more efficient photosynthetic electron transport compared to the wild type. However, all phot mutants exhibited higher levels of cell death four days after UV-C treatment, as well as deregulated gene expression. Taken together, our results reveal that on the one hand, both phot1 and phot2 contribute to the inhibition of UV-C-induced foliar cell death, but on the other hand, they also contribute to the maintenance of foliar H2O2 levels and optimal intensity of photochemical reactions and non-photochemical quenching after an exposure to UV-C stress. Our data indicate a novel role for phototropins in the condition-dependent optimization of photosynthesis, growth, and water-use efficiency as well as oxidative stress and cell death response after UV-C exposure.

EDS1-Dependent Cell Death and the Antioxidant System in Arabidopsis Leaves is Deregulated by the Mammalian Bax.

Cell death is the ultimate end of a cell cycle that occurs in all living organisms during development or responses to biotic and abiotic stresses. In the course of evolution, plants and animals evolve various molecular mechanisms to regulate cell death; however, some of them are conserved among both these kingdoms. It was found that mammalian proapoptotic BCL-2 associated X (Bax) protein, when expressed in plants, induces cell death, similar to hypersensitive response (HR). It was also shown that changes in the expression level of genes encoding proteins involved in stress response or oxidative status regulation mitigate Bax-induced plant cell death. In our study, we focused on the evolutional compatibility of animal and plant cell death molecular mechanisms. Therefore, we studied the deregulation of reactive oxygen species burst and HR-like propagation in Arabidopsis thaliana expressing mammalian Bax. We were able to diminish Bax-induced oxidative stress and HR progression through the genetic cross with plants mutated in ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), which is a plant-positive HR regulator. Plants expressing the mouse Bax gene in eds1-1 null mutant background demonstrated less pronounced cell death and exhibited higher antioxidant system efficiency compared to Bax-expressing plants. Moreover, eds1/Bax plants did not show HR marker genes induction, as in the case of the Bax-expressing line. The present study indicates some common molecular features between animal and plant cell death regulation and can be useful to better understand the evolution of cell death mechanisms in plants and animals.

FMO1 Is Involved in Excess Light Stress-Induced Signal Transduction and Cell Death Signaling.

Because of their sessile nature, plants evolved integrated defense and acclimation mechanisms to simultaneously cope with adverse biotic and abiotic conditions. Among these are systemic acquired resistance (SAR) and systemic acquired acclimation (SAA). Growing evidence suggests that SAR and SAA activate similar cellular mechanisms and employ common signaling pathways for the induction of acclimatory and defense responses. It is therefore possible to consider these processes together, rather than separately, as a common systemic acquired acclimation and resistance (SAAR) mechanism. Arabidopsis thaliana flavin-dependent monooxygenase 1 (FMO1) was previously described as a regulator of plant resistance in response to pathogens as an important component of SAR. In the current study, we investigated its role in SAA, induced by a partial exposure of Arabidopsis rosette to local excess light stress. We demonstrate here that FMO1 expression is induced in leaves directly exposed to excess light stress as well as in systemic leaves remaining in low light. We also show that FMO1 is required for the systemic induction of ASCORBATE PEROXIDASE 2 (APX2) and ZINC-FINGER OF ARABIDOPSIS 10 (ZAT10) expression and spread of the reactive oxygen species (ROS) systemic signal in response to a local application of excess light treatment. Additionally, our results demonstrate that FMO1 is involved in the regulation of excess light-triggered systemic cell death, which is under control of LESION SIMULATING DISEASE 1 (LSD1). Our study indicates therefore that FMO1 plays an important role in triggering SAA response, supporting the hypothesis that SAA and SAR are tightly connected and use the same signaling pathways.

Novel Role of JAC1 in Influencing Photosynthesis, Stomatal Conductance, and Photooxidative Stress Signalling Pathway in Arabidopsis thaliana.

Regulation of light absorption under variable light conditions is essential to optimize photosynthetic and acclimatory processes in plants. Light energy absorbed in excess has a damaging effect on chloroplasts and can lead to cell death. Therefore, plants have evolved protective mechanisms against excess excitation energy that include chloroplast accumulation and avoidance responses. One of the proteins involved in facilitating chloroplast movements in Arabidopsis thaliana is the J domain-containing protein required for chloroplast accumulation response 1 (JAC1). The function of JAC1 relates to the chloroplast actin filaments appearance and disappearance. So far, the role of JAC1 was studied mainly in terms of chloroplasts photorelocation. Here, we demonstrate that the function of JAC1 is more complex, since it influences the composition of photosynthetic pigments, the efficiency of photosynthesis, and the CO2 uptake rate. JAC1 has positive effect on water use efficiency (WUE) by reducing stomatal aperture and water vapor conductance. Importantly, we show that the stomatal aperture regulation is genetically coupled with JAC1 activity. In addition, our data demonstrate that JAC1 is involved in the fine-tuning of H2O2 foliar levels, antioxidant enzymes activities and cell death after UV-C photooxidative stress. This work uncovers a novel function for JAC1 in affecting photosynthesis, CO2 uptake, and photooxidative stress responses.

Biotechnological Potential of LSD1, EDS1, and PAD4 in the Improvement of Crops and Industrial Plants.

Lesion Simulating Disease 1 (LSD1), Enhanced Disease Susceptibility (EDS1) and Phytoalexin Deficient 4 (PAD4) were discovered a quarter century ago as regulators of programmed cell death and biotic stress responses in Arabidopsis thaliana. Recent studies have demonstrated that these proteins are also required for acclimation responses to various abiotic stresses, such as high light, UV radiation, drought and cold, and that their function is mediated through secondary messengers, such as salicylic acid (SA), reactive oxygen species (ROS), ethylene (ET) and other signaling molecules. Furthermore, LSD1, EDS1 and PAD4 were recently shown to be involved in the modification of cell walls, and the regulation of seed yield, biomass production and water use efficiency. The function of these proteins was not only demonstrated in model plants, such as Arabidopsis thaliana or Nicotiana benthamiana, but also in the woody plant Populus tremula x tremuloides. In addition, orthologs of LSD1, EDS1, and PAD4 were found in other plant species, including different crop species. In this review, we focus on specific LSD1, EDS1 and PAD4 features that make them potentially important for agricultural and industrial use.

Transcriptomic Changes in Medicago truncatula and Lotus japonicus Root Nodules during Drought Stress.

Drought is one of the major environmental factors limiting biomass and seed yield production in agriculture. In this research, we focused on plants from the Fabaceae family, which has a unique ability for the establishment of symbiosis with nitrogen-fixing bacteria, and are relatively susceptible to water limitation. We have presented the changes in nitrogenase activity and global gene expression occurring in Medicago truncatula and Lotus japonicus root nodules during water deficit. Our results proved a decrease in the efficiency of nitrogen fixation, as well as extensive changes in plant and bacterial transcriptomes, shortly after watering cessation. We showed for the first time that not only symbiotic plant components but also Sinorhizobium meliloti and Mesorhizobium loti bacteria residing in the root nodules of M. truncatula and L. japonicus, respectively, adjust their gene expression in response to water shortage. Although our results demonstrated that both M. truncatula and L. japonicus root nodules were susceptible to water deprivation, they indicated significant differences in plant and bacterial response to drought between the tested species, which might be related to the various types of root nodules formed by these species.

Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus.

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical ß-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root's stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

LSD1-, EDS1- and PAD4-dependent conditional correlation among salicylic acid, hydrogen peroxide, water use efficiency and seed yield in Arabidopsis thaliana.

In Arabidopsis thaliana, LESION SIMULATING DISEASE 1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) proteins are regulators of cell death (CD) in response to abiotic and biotic stresses. Hormones, such as salicylic acid (SA), and reactive oxygen species, such as hydrogen peroxide (H2 O2 ), are key signaling molecules involved in plant CD. The proposed mathematical models presented in this study suggest that LSD1, EDS1 and PAD4 together with SA and H2 O2 are involved in the control of plant water use efficiency (WUE), vegetative growth and generative development. The analysis of Arabidopsis wild-type and single mutants lsd1, eds1, and pad4, as well as double mutants eds1/lsd1 and pad4/lsd1, demonstrated the strong conditional correlation between SA/H2 O2 and WUE that is dependent on LSD1, EDS1 and PAD4 proteins. Moreover, we found a strong correlation between the SA/H2 O2 homeostasis of 4-week-old Arabidopsis leaves and a total seed yield of 9-week-old plants. Altogether, our results prove that SA and H2 O2 are conditionally regulated by LSD1/EDS/PAD4 to govern WUE, biomass accumulation and seed yield. Conditional correlation and the proposed models presented in this study can be used as the starting points in the creation of a plant breeding algorithm that would allow to estimate the seed yield at the initial stage of plant growth, based on WUE, SA and H2 O2 content.

Expression of both Arabidopsis γ-tubulin genes is essential for development of a functional syncytium induced by Heterodera schachtii.

KEY MESSAGE: After initial up-regulation, expression of TUBG1 and TUBG2 is significantly down-regulated in mature syncytia, but lack of expression of either of γ-tubulin genes reduces numbers of nematode infections and developing females. Infective second stage juveniles of sedentary plant parasitic nematode Heterodera schachtii invade the root vascular tissue and induce a feeding site, named syncytium, formed as a result of cell hypertrophy and partial cell wall dissolution leading to a multinucleate state. Syncytium formation and maintenance involves a molecular interplay between the plant host and the developing juveniles leading to rearrangements and fragmentation of the plant cytoskeleton. In this study, we investigated the role of two Arabidopsis γ-tubulin genes (TUBG1 and TUBG2), involved in MTs nucleation during syncytium development. Expression analysis revealed that both γ-tubulin's transcript levels changed during syncytium development and after initial up-regulation (1-3 dpi) they were significantly down-regulated in 7, 10 and 15 dpi syncytia. Moreover, TUBG1 and TUBG2 showed distinct immunolocalization patterns in uninfected roots and syncytia. Although no severe changes in syncytium anatomy and ultrastructure in tubg1-1 and tubg2-1 mutants were observed compared to syncytia induced in wild-type plants, nematode infection assays revealed reduced numbers of infecting juveniles and developed female nematodes in mutant lines. Our results indicate that the expression of both TUBG1 and TUBG2 genes, although generally down-regulated in mature syncytia, is essential for successful root infection, development of functional syncytium and nematode maturation.

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) affects development, photosynthesis, and hormonal homeostasis in hybrid aspen (Populus tremula L. × P. tremuloides).

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) was first described as a protein involved in salicylic acid (SA)-, ethylene-, and reactive oxygen species (ROS)-dependent defense and acclimation responses. It is a molecular regulator of biotic and abiotic stress-induced programmed cell death. Its role is relatively well known in annual plants, such as Arabidopsis thaliana or Nicotiana benthamiana. However, little is known about its functions in woody plants. Therefore, in this study, we aimed to characterize the function of EDS1 in the Populus tremula L. × P. tremuloides hybrid grown for several seasons in the natural environment. We used two transgenic lines, eds1-7 and eds1-12, with decreased EDS1 expression levels in this study. The observed changes in physiological and biochemical parameters corresponded with the EDS1 silencing level. Both transgenic lines produced more lateral shoots in comparison to the wild-type (WT) plants, which resulted in the modification of tree morphology. Photosynthetic parameters, such as quantum yield of photosystem II (ϕPSII), photochemical and non-photochemical quenching (qP and NPQ, respectively), as well as chlorophyll content were found to be increased in both transgenic lines, which resulted in changes in photosynthetic efficiency. Our data also revealed lower foliar concentrations of SA and ROS, the latter resulting most probably from more efficient antioxidant system in both transgenic lines. In addition, our data indicated significantly decreased rate of leaf senescence during several autumn seasons. Transcriptomic analysis revealed deregulation of 2215 and 376 genes in eds1-12 and eds1-7, respectively, and also revealed 207 genes that were commonly deregulated in both transgenic lines. The deregulation was primarily observed in the genes involved in photosynthesis, signaling, hormonal metabolism, and development, which was found to agree with the results of biochemical and physiological tests. In general, our data proved that poplar EDS1 affects tree morphology, photosynthetic efficiency, ROS and SA metabolism, as well as leaf senescence.

Friend or foe? Reactive oxygen species production, scavenging and signaling in plant response to environmental stresses.

In the natural environment, plants are exposed to a variety of biotic and abiotic stress conditions that trigger rapid changes in the production and scavenging of reactive oxygen species (ROS). The production and scavenging of ROS is compartmentalized, which means that, depending on stimuli type, they can be generated and eliminated in different cellular compartments such as the apoplast, plasma membrane, chloroplasts, mitochondria, peroxisomes, and endoplasmic reticulum. Although the accumulation of ROS is generally harmful to cells, ROS play an important role in signaling pathways that regulate acclimatory and defense responses in plants, such as systemic acquired acclimation (SAA) and systemic acquired resistance (SAR). However, high accumulations of ROS can also trigger redox homeostasis disturbance which can lead to cell death, and in consequence, to a limitation in biomass and yield production. Different ROS have various half-lifetimes and degrees of reactivity toward molecular components such as lipids, proteins, and nucleic acids. Thus, they play different roles in intra- and extra-cellular signaling. Despite their possible damaging effect, ROS should mainly be considered as signaling molecules that regulate local and systemic acclimatory and defense responses. Over the past two decades it has been proven that ROS together with non-photochemical quenching (NPQ), hormones, Ca2+ waves, and electrical signals are the main players in SAA and SAR, two physiological processes essential for plant survival and productivity in unfavorable conditions.

Effect of short-term aluminum stress and mycorrhizal inoculation on nitric oxide metabolism in Medicago truncatula roots.

Aluminum (Al) toxicity can induce oxidative and nitrosative stress, which limits growth and yield of crop plants. Nevertheless, plant tolerance to stress may be improved by symbiotic associations including arbuscular mycorrhiza (AM). Nitric oxide (NO) is a signaling molecule involved in physiological processes and plant responses to abiotic and biotic stresses. However, almost no information about the NO metabolism has been gathered about AM. In the present work, Medicago truncatula seedlings were inoculated with Rhizophagus irregularis, and 7-week-old plants were treated with 50µM AlCl3 for 3h. Cytochemical and molecular techniques were used to measure the components of the NO metabolism, including NO content and localization, expression of genes encoding NO-synthesis (MtNR1, MtNR2 and MtNIR1) and NO-scavenging (MtGSNOR1, MtGSNOR2, MtHB1 and MtHB2) enzymes and the profile of protein tyrosine nitration (NO2-Tyr) in Medicago roots. For the first time, NO and NO2-Tyr accumulation was connected with fungal structures (arbuscules, vesicles and intercellular hyphae). Expression analysis of genes encoding NO-synthesis enzymes indicated that AM symbiosis results in lower production of NO in Al-treated roots in comparison to non-mycorrhizal roots. Elevated levels of transcription of genes encoding NO-scavenging enzymes indicated more active NO scavenging in AMF-inoculated Al-treated roots compared to non-inoculated roots. These results were confirmed by less NO accumulation and lower protein nitration in Al-stressed mycorrhizal roots in comparison to non-mycorrhizal roots. This study provides a new insight in NO metabolism in response to arbuscular mycorrhiza under normal and metal stress conditions. Our results suggest that mycorrhizal fungi decrease NO and tyrosine nitrated proteins content in Al-treated Medicago roots, probably via active NO scavenging system.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii.

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

The dual role of LESION SIMULATING DISEASE 1 as a condition-dependent scaffold protein and transcription regulator.

Since its discovery over two decades ago as an important cell death regulator in Arabidopsis thaliana, the role of LESION SIMULATING DISEASE 1 (LSD1) has been studied intensively within both biotic and abiotic stress responses as well as with respect to plant fitness regulation. However, its molecular mode of action remains enigmatic. Here, we demonstrate that nucleo-cytoplasmic LSD1 interacts with a broad range of other proteins that are engaged in various molecular pathways such as ubiquitination, methylation, cell cycle control, gametogenesis, embryo development and cell wall formation. The interaction of LSD1 with these partners is dependent on redox status, as oxidative stress significantly changes the quantity and types of LSD1-formed complexes. Furthermore, we show that LSD1 regulates the number and size of leaf mesophyll cells and affects plant vegetative growth. Importantly, we also reveal that in addition to its function as a scaffold protein, LSD1 acts as a transcriptional regulator. Taken together, our results demonstrate that LSD1 plays a dual role within the cell by acting as a condition-dependent scaffold protein and as a transcription regulator.

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula.

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA(-)). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1-MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.