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.

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.

Photosystem II 22kDa protein level - a prerequisite for excess light-inducible memory, cross-tolerance to UV-C and regulation of electrical signalling.

It is well known that PsbS is a key protein for the proper management of excessive energy in plants. Plants without PsbS cannot trigger non-photochemical quenching, which is crucial for optimal photosynthesis under variable conditions. Our studies showed wild-type plants had enhanced tolerance to UV-C-induced cell death (CD) upon induction of light memory by a blue or red light. However, npq4-1 plants, which lack PsbS, as well as plants overexpressing this protein (oePsbS), responded differently. Untreated oePsbS appeared more tolerant to UV-C exposure, whereas npq4-1 was unable to adequately induce cross-tolerance to UV-C. Similarly, light memory induced by episodic blue or red light was differently deregulated in npq-4 and oePsbS, as indicated by transcriptomic analyses, measurements of the trans-thylakoid pH gradient, chlorophyll a fluorescence parameters, and measurements of foliar surface electrical potential. The mechanism of the foliar CD development seemed to be unaffected in the analysed plants and is associated with chloroplast breakdown. Our results suggest a novel, substantial role for PsbS as a regulator of chloroplast retrograde signalling for light memory, light acclimation, CD, and cross-tolerance to UV radiation.

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.

The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs.

Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism.

ß-carbonic anhydrases and carbonic ions uptake positively influence Arabidopsis photosynthesis, oxidative stress tolerance and growth in light dependent manner.

Carbonic anhydrases (CAs) catalyse reversible interconversion of CO2 and water into bicarbonate and protons and regulate concentration of CO2 around photosynthetic enzymes. In higher plants the CAs are divided into three distinct classes α, ß and γ, with members off each of them being involved in CO2 uptake, fixation or recycling. The most abundant group is ßCAs. In C4 plants they are localized in the cytosol of mesophyll cells and catalyse first step of carbon concentration pathway. C3 plants contain orthologues genes encoding ßCAs's, however their functions are unknown. Given the importance of ßCAs in the present study we analysed the effect of carbonic ions, selected orthologues ßCAs's gene expression and ßCAs enzymatic activity on Arabidopsis photosynthesis, growth and cell death in different light conditions. Plants fertilised with 0.5-3mM sodium bicarbonate had a significantly increased number of leaves, improved fresh and dry weight and reduced cell death (cellular ion leakage). This effect was dependent on provided photon flux density and photoperiod. Higher content of carbonic ions also stimulated photoprotective mechanisms such as non-photochemical quenching and foliar content of photoprotective pigments (neoxanthin, violaxanthin and carotenes). Function of various ßCAs genes examined in null ßcas mutants showed to be complementary and additive, and confirm results of fertilizing experiments. Taken together, regulation of ßCAs gene expression and enzymatic activities are important for optimal plant growth and probably can be one of the factor influencing a switch between C3 and C4 photosynthesis mode in variable light conditions. Therefore, biotechnological amelioration of ßCAs activity in economically important plants and their fertilisation with carbonic ions may lead to improved photosynthetic efficiency and further crop productivity.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

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.