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.

The CRK5 and WRKY53 Are Conditional Regulators of Senescence and Stomatal Conductance in Arabidopsis.

In Arabidopsis thaliana, cysteine-rich receptor-like kinases (CRKs) constitute a large group of membrane-localized proteins which perceive external stimuli and transduce the signal into the cell. Previous reports based on their loss-of-function phenotypes and expression profile support their role in many developmental and stress-responsive pathways. Our study revealed that one member of this family, CRK5, acts as a negative regulator of leaf aging. Enrichment of the CRK5 promoter region in W-box cis-elements demonstrated that WRKY transcription factors control it. We observed significantly enhanced WRKY53 expression in crk5 and reversion of its early-senescence phenotype in the crk5 wrky53 line, suggesting a negative feedback loop between these proteins antagonistically regulating chlorophyll a and b contents. Yeast-two hybrid assay showed further that CRK5 interacts with several proteins involved in response to water deprivation or calcium signaling, while gas exchange analysis revealed a positive effect of CRK5 on water use efficiency. Consistent with that, the crk5 plants showed disturbed foliar temperature, stomatal conductance, transpiration, and increased susceptibility to osmotic stress. These traits were fully or partially reverted to wild-type phenotype in crk5 wrky53 double mutant. Obtained results suggest that WRKY53 and CRK5 are antagonistic regulators of chlorophyll synthesis/degradation, senescence, and stomatal conductance.

Aux/IAA11 Is Required for UV-AB Tolerance and Auxin Sensing in Arabidopsis thaliana

In order to survive, plants have, over the course of their evolution, developed sophisticated acclimation and defense strategies governed by complex molecular and physiological, and cellular and extracellular, signaling pathways. They are also able to respond to various stimuli in the form of tropisms; for example, phototropism or gravitropism. All of these retrograde and anterograde signaling pathways are controlled and regulated by waves of reactive oxygen species (ROS), electrical signals, calcium, and hormones, e.g., auxins. Auxins are key phytohormones involved in the regulation of plant growth and development. Acclimation responses, which include programmed cell death induction, require precise auxin perception. However, our knowledge of these pathways is limited. The Aux/IAA family of transcriptional corepressors inhibits the growth of the plant under stress conditions, in order to maintain the balance between development and acclimation responses. In this work, we demonstrate the Aux/IAA11 involvement in auxin sensing, survival, and acclimation to UV-AB, and in carrying out photosynthesis under inhibitory conditions. The tested iaa11 mutants were more susceptible to UV-AB, photosynthetic electron transport (PET) inhibitor, and synthetic endogenous auxin. Among the tested conditions, Aux/IAA11 was not repressed by excess light stress, exclusively among its phylogenetic clade. Repression of transcription by Aux/IAA11 could be important for the inhibition of ROS formation or efficiency of ROS scavenging. We also hypothesize that the demonstrated differences in the subcellular localization of the two Aux/IAA11 protein variants might indicate their regulation by alternative splicing. Our results suggest that Aux/IAA11 plays a specific role in chloroplast retrograde signaling, since it is not repressed by high (excess) light stress, exclusively among its phylogenetic clade.

CIA2 and CIA2-LIKE are required for optimal photosynthesis and stress responses in Arabidopsis thaliana.

Chloroplast-to-nucleus retrograde signaling is essential for cell function, acclimation to fluctuating environmental conditions, plant growth and development. The vast majority of chloroplast proteins are nuclear-encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses that influence nuclear gene expression. Signaling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual-localized TFs involved in chloroplast retrograde signaling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homolog CIA2-like (CIL) are involved in the regulation of Arabidopsis responses to UV-AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signaling events in the chloroplast-nucleus cross-talk.

Retrograde Signaling: Understanding the Communication between Organelles.

Understanding how cell organelles and compartments communicate with each other has always been an important field of knowledge widely explored by many researchers. However, despite years of investigations, one point-and perhaps the only point that many agree on-is that our knowledge about cellular-signaling pathways still requires expanding. Chloroplasts and mitochondria (because of their primary functions in energy conversion) are important cellular sensors of environmental fluctuations and feedback they provide back to the nucleus is important for acclimatory responses. Under stressful conditions, it is important to manage cellular resources more efficiently in order to maintain a proper balance between development, growth and stress responses. For example, it can be achieved through regulation of nuclear and organellar gene expression. If plants are unable to adapt to stressful conditions, they will be unable to efficiently produce energy for growth and development-and ultimately die. In this review, we show the importance of retrograde signaling in stress responses, including the induction of cell death and in organelle biogenesis. The complexity of these pathways demonstrates how challenging it is to expand the existing knowledge. However, understanding this sophisticated communication may be important to develop new strategies of how to improve adaptability of plants in rapidly changing environments.

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.

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 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.