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.

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.

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 Role of Programmed Cell Death Regulator LSD1 in Nematode-Induced Syncytium Formation.

Cyst-forming plant-parasitic nematodes are common pests of many crops. They inject secretions into host cells to induce the developmental and metabolic reprogramming that leads to the formation of a syncytium, which is the sole food source for growing nematodes. As in other host-parasite models, avirulence leads to rapid and local programmed cell death (PCD) known as the hypersensitive response (HR), whereas in the case of virulence, PCD is still observed but is limited to only some cells. Several regulators of PCD were analyzed to understand the role of PCD in compatible plant-nematode interactions. Thus, Arabidopsis plants carrying recessive mutations in LESION SIMULATING DISEASE1 (LSD1) family genes were subjected to nematode infection assays with juveniles of Heterodera schachtii. LSD1 is a negative and conditional regulator of PCD, and fewer and smaller syncytia were induced in the roots of lsd1 mutants than in wild-type Col-0 plants. Mutation in LSD ONE LIKE2 (LOL2) revealed a pattern of susceptibility to H. schachtii antagonistic to lsd1. Syncytia induced on lsd1 roots compared to Col0 showed significantly retarded growth, modified cell wall structure, increased vesiculation, and some myelin-like bodies present at 7 and 12 days post-infection. To place these data in a wider context, RNA-sequencing analysis of infected and uninfected roots was conducted. During nematode infection, the number of transcripts with changed expression in lsd1 was approximately three times smaller than in wild-type plants (1440 vs. 4206 differentially expressed genes, respectively). LSD1-dependent PCD in roots is thus a highly regulated process in compatible plant-nematode interactions. Two genes identified in this analysis, coding for AUTOPHAGY-RELATED PROTEIN 8F and 8H were down-regulated in syncytia in the presence of LSD1 and showed an increased susceptibility to nematode infection contrasting with lsd1 phenotype. Our data indicate that molecular regulators belonging to the LSD1 family play an important role in precise balancing of diverse PCD players during syncytium development required for successful nematode parasitism.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

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.

Evidence for light wavelength-specific photoelectrophysiological signaling and memory of excess light episodes in Arabidopsis.

Although light is essential for photosynthesis, excess light can damage the photosynthetic apparatus and deregulate other cellular processes. Thus, protective integrated regulatory responses that can dissipate excess of absorbed light energy and simultaneously optimize photosynthesis and other cellular processes under variable light conditions can prove highly adaptive. Here, we show that the local and systemic responses to an excess light episode are associated with photoelectrophysiological signaling (PEPS) as well as with changes in nonphotochemical quenching and reactive oxygen species levels. During an excess light incident, PEPS is induced by quantum redox changes in photosystem II and in its proximity and/or by changes in glutathione metabolism in chloroplasts. PEPS is transduced, at least in part, by bundle sheath cells and is light wavelength specific. PEPS systemic propagation speed and action potential are dependent on ASCORBATE PEROXIDASE2 function. Excess light episodes are physiologically memorized in leaves, and the cellular light memory effect is specific for an excess of blue (450 nm) and red (650 nm) light of similar energy. It is concluded that plants possess a complex and dynamic light training and memory system that involves quantum redox, reactive oxygen species, hormonal, and PEPS signaling and is used to optimize light acclimation and immune defenses.

Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis.

BACKGROUND: Plant Receptor-like/Pelle kinases (RLK) are a group of conserved signalling components that regulate developmental programs and responses to biotic and abiotic stresses. One of the largest RLK groups is formed by the Domain of Unknown Function 26 (DUF26) RLKs, also called Cysteine-rich Receptor-like Kinases (CRKs), which have been suggested to play important roles in the regulation of pathogen defence and programmed cell death. Despite the vast number of RLKs present in plants, however, only a few of them have been functionally characterized. RESULTS: We examined the transcriptional regulation of all Arabidopsis CRKs by ozone (O3), high light and pathogen/elicitor treatment - conditions known to induce the production of reactive oxygen species (ROS) in various subcellular compartments. Several CRKs were transcriptionally induced by exposure to O3 but not by light stress. O3 induces an extracellular oxidative burst, whilst light stress leads to ROS production in chloroplasts. Analysis of publicly available microarray data revealed that the transcriptional responses of the CRKs to O3 were very similar to responses to microbes or pathogen-associated molecular patterns (PAMPs). Several mutants altered in hormone biosynthesis or signalling showed changes in basal and O3-induced transcriptional responses. CONCLUSIONS: Combining expression analysis from multiple treatments with mutants altered in hormone biosynthesis or signalling suggest a model in which O3 and salicylic acid (SA) activate separate signaling pathways that exhibit negative crosstalk. Although O3 is classified as an abiotic stress to plants, transcriptional profiling of CRKs showed strong similarities between the O3 and biotic stress responses.

Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis.

Sudden exposure of plants to high light (HL) leads to metabolic and physiological disruption of the photosynthetic cells. Changes in ROS content, adjustment of photosynthetic processes and the antioxidant pools and, ultimately, gene induction are essential components for a successful acclimation to the new light conditions. The influence of salicylic acid (SA) on plant growth, short-term acclimation to HL, and on the redox homeostasis of Arabidopsis thaliana leaves was assessed here. The dwarf phenotype displayed by mutants with high SA content (cpr1-1, cpr5-1, cpr6-1, and dnd1-1) was less pronounced when these plants were grown in HL, suggesting that the inhibitory effect of SA on growth was partly overcome at higher light intensities. Moreover, higher SA content affected energy conversion processes in low light, but did not impair short-term acclimation to HL. On the other hand, mutants with low foliar SA content (NahG and sid2-2) were impaired in acclimation to transient exposure to HL and thus predisposed to oxidative stress. Low and high SA levels were strictly correlated to a lower and higher foliar H(2)O(2) content, respectively. Furthermore high SA was also associated with higher GSH contents, suggesting a tight correlation between SA, H(2)O(2) and GSH contents in plants. These observations implied an essential role of SA in the acclimation processes and in regulating the redox homeostasis of the cell. Implications for the role of SA in pathogen defence signalling are also discussed.

Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis.

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had >/=50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of gamma-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic gamma-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum L.

A strongly increased ATP/ADP ratio was found during the nocturnal phase I in crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants. Conversely, during the daytime phase III in CAM-performing plants the ATP/ADP ratio dropped to a similar level to that of C3 plants, cytochrome c oxidase activity was stimulated and mitochondrial Mn-superoxide dismutase activity was strongly increased. The findings suggest that a salinity-induced C3-CAM transition might be an efficient energy-conserving strategy for M. crystallinum plants, in which the strong nocturnal ATP production seems to be, at least partially, independent from the coupled mitochondrial electron transport.