Biotechnological Potential of the Stress Response and Plant Cell Death Regulators Proteins in the Biofuel Industry.

Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death-a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. "Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.

Aboveground plant-to-plant electrical signaling mediates network acquired acclimation.

Systemic acquired acclimation and wound signaling require the transmission of electrical, calcium, and reactive oxygen species (ROS) signals between local and systemic tissues of the same plant. However, whether such signals can be transmitted between two different plants is largely unknown. Here, we reveal a new type of plant-to-plant aboveground direct communication involving electrical signaling detected at the surface of leaves, ROS, and photosystem networks. A foliar electrical signal induced by wounding or high light stress applied to a single dandelion leaf can be transmitted to a neighboring plant that is in direct contact with the stimulated plant, resulting in systemic photosynthetic, oxidative, molecular, and physiological changes in both plants. Furthermore, similar aboveground changes can be induced in a network of plants serially connected via touch. Such signals can also induce responses even if the neighboring plant is from a different plant species. Our study demonstrates that electrical signals can function as a communication link between transmitter and receiver plants that are organized as a network (community) of plants. This process can be described as network-acquired acclimation.

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.

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.

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.

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.

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.

Contribution of PsbS Function and Stomatal Conductance to Foliar Temperature in Higher Plants.

Natural capacity has evolved in higher plants to absorb and harness excessive light energy. In basic models, the majority of absorbed photon energy is radiated back as fluorescence and heat. For years the proton sensor protein PsbS was considered to play a critical role in non-photochemical quenching (NPQ) of light absorbed by PSII antennae and in its dissipation as heat. However, the significance of PsbS in regulating heat emission from a whole leaf has never been verified before by direct measurement of foliar temperature under changing light intensity. To test its validity, we here investigated the foliar temperature changes on increasing and decreasing light intensity conditions (foliar temperature dynamics) using a high resolution thermal camera and a powerful adjustable light-emitting diode (LED) light source. First, we showed that light-dependent foliar temperature dynamics is correlated with Chl content in leaves of various plant species. Secondly, we compared the foliar temperature dynamics in Arabidopsis thaliana wild type, the PsbS null mutant npq4-1 and a PsbS-overexpressing transgenic line under different transpiration conditions with or without a photosynthesis inhibitor. We found no direct correlations between the NPQ level and the foliar temperature dynamics. Rather, differences in foliar temperature dynamics are primarily affected by stomatal aperture, and rapid foliar temperature increase during irradiation depends on the water status of the leaf. We conclude that PsbS is not directly involved in regulation of foliar temperature dynamics during excessive light energy episodes.

Role of phytochromes A and B in the regulation of cell death and acclimatory responses to UV stress in Arabidopsis thaliana.

Plants coordinate their responses to various biotic and abiotic stresses in order to optimize their developmental and acclimatory programmes. The ultimate response to an excessive amount of stress is local induction of cell death mechanisms. The death of certain cells can help to maintain tissue homeostasis and enable nutrient remobilization, thus increasing the survival chances of the whole organism in unfavourable environmental conditions. UV radiation is one of the environmental factors that negatively affects the photosynthetic process and triggers cell death. The aim of this work was to evaluate a possible role of the red/far-red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) and their interrelations during acclimatory responses to UV stress. We showed that UV-C treatment caused a disturbance in photosystem II and a deregulation of photosynthetic pigment content and antioxidant enzymes activities, followed by increased cell mortality rate in phyB and phyAB null mutants. We also propose a regulatory role of phyA and phyB in CO2 assimilation, non-photochemical quenching, reactive oxygen species accumulation and salicylic acid content. Taken together, our results suggest a novel role of phytochromes as putative regulators of cell death and acclimatory responses to UV.

Cysteine-rich receptor-like kinase CRK5 as a regulator of growth, development, and ultraviolet radiation responses in Arabidopsis thaliana.

In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death and oxidative damage in response to ultraviolet radiation. Complementation of CRK5 mutation managed to recover the wild-type phenotype, indicating an essential role of this gene in the regulation of growth, development, and acclimatory responses.

PHYTOALEXIN DEFICIENT 4 affects reactive oxygen species metabolism, cell wall and wood properties in hybrid aspen (Populus tremula L. × tremuloides).

The phytoalexin deficient 4 (PAD4) gene in Arabidopsis thaliana (AtPAD4) is involved in the regulation of plant--pathogen interactions. The role of PAD4 in woody plants is not known; therefore, we characterized its function in hybrid aspen and its role in reactive oxygen species (ROS)-dependent signalling and wood development. Three independent transgenic lines with different suppression levels of poplar PAD expression were generated. All these lines displayed deregulated ROS metabolism, which was manifested by an increased H2O2 level in the leaves and shoots, and higher activities of manganese superoxide dismutase (MnSOD) and catalase (CAT) in the leaves in comparison to the wild-type plants. However, no changes in non-photochemical quenching (NPQ) between the transgenic lines and wild type were observed in the leaves. Moreover, changes in the ROS metabolism in the pad4 transgenic lines positively correlated with wood formation. A higher rate of cell division, decreased tracheid average size and numbers, and increased cell wall thickness were observed. The results presented here suggest that the Populus tremula × tremuloides PAD gene might be involved in the regulation of cellular ROS homeostasis and in the cell division--cell death balance that is associated with wood development.

Lesion simulating disease 1 and enhanced disease susceptibility 1 differentially regulate UV-C-induced photooxidative stress signalling and programmed cell death in Arabidopsis thaliana.

As obligate photoautotrophs, plants are inevitably exposed to ultraviolet (UV) radiation. Because of stratospheric ozone depletion, UV has become more and more dangerous to the biosphere. Therefore, it is important to understand UV perception and signal transduction in plants. In the present study, we show that lesion simulating disease 1 (LSD1) and enhanced disease susceptibility 1 (EDS1) are antagonistic regulators of UV-C-induced programmed cell death (PCD) in Arabidopsis thaliana. This regulatory dependence is manifested by a complex deregulation of photosynthesis, reactive oxygen species homeostasis, antioxidative enzyme activity and UV-responsive genes expression. We also prove that a UV-C radiation episode triggers apoptotic-like morphological changes within the mesophyll cells. Interestingly, chloroplasts are the first organelles that show features of UV-C-induced damage, which may indicate their primary role in PCD development. Moreover, we show that Arabidopsis Bax inhibitor 1 (AtBI1), which has been described as a negative regulator of plant PCD, is involved in LSD1-dependent cell death in response to UV-C. Our results imply that LSD1 and EDS1 regulate processes extinguishing excessive energy, reactive oxygen species formation and subsequent PCD in response to different stresses related to impaired electron transport.

Mitogen-activated protein kinase 4 is a salicylic acid-independent regulator of growth but not of photosynthesis in Arabidopsis.

Mitogen-activated protein kinase (MAPK) pathways regulate signal transduction from different cellular compartments and from the extracellular environment to the nucleus in all eukaryotes. One of the best-characterized MAPKs in Arabidopsis thaliana is MPK4, which was shown to be a negative regulator of systemic-acquired resistance. The mpk4 mutant accumulates salicylic acid (SA), possesses constitutive expression of pathogenesis-related (PR) genes, and has an extremely dwarf phenotype. We show that suppression of SA and phylloquinone synthesis in chloroplasts by knocking down the ICS1 gene (by crossing it with the ics1 mutant) in the mpk4 mutant background did not revert mpk4-impaired growth. However, it did cause changes in the photosynthetic apparatus and severely impaired the quantum yield of photosystem II. Transmission microscopy analysis revealed that the chloroplasts' structure was strongly altered in the mpk4 and mpk4/ics1 double mutant. Analysis of reactive oxygen species (ROS)-scavenging enzymes expression showed that suppression of SA and phylloquinone synthesis in the chloroplasts of the mpk4 mutant caused imbalances in ROS homeostasis which were more pronounced in mpk4/ics1 than in mpk4. Taken together, the presented results strongly suggest that MPK4 is an ROS/hormonal rheostat hub that negatively, in an SA-dependent manner, regulates immune defenses, but at the same time positively regulates photosynthesis, ROS metabolism, and growth. Therefore, we concluded that MPK4 is a complex regulator of chloroplastic retrograde signaling for photosynthesis, growth, and immune defenses in Arabidopsis.