CAD1 contributes to osmotic tolerance in Arabidopsis thaliana by suppressing immune responses under osmotic stress.

Acquired osmotolerance induced by initial exposure to mild salt stress is widespread across Arabidopsis thaliana ecotypes, but the mechanism underlying it remains poorly understood. To clarify it, we isolated acquired osmotolerance-deficient 1 (aod1), a mutant highly sensitive to osmotic stress, from ion-beam-irradiated seeds of Zu-0, an ecotype known for its remarkably high osmotolerance. Aod1 showed growth inhibition with spotted necrotic lesions on the rosette leaves under normal growth conditions on soil. However, its tolerance to salt and oxidative stresses was similar to that of the wild type (WT). Genetic and genome sequencing analyses suggested that the gene causing aod1 is identical to CONSTITUTIVELY ACTIVATED CELL DEATH 1 (CAD1). Complementation with the WT CAD1 gene restored the growth and osmotolerance of aod1, indicating that mutated CAD1 is responsible for the observed phenotypes in aod1. Although CAD1 is known to act as a negative regulator of immune response, transcript levels in the WT increased in response to osmotic stress. Aod1 displayed enhanced immune response and cell death under normal growth conditions, whereas the expression profiles of osmotic response genes were comparable to those of the WT. These findings suggest that autoimmunity in aod1 is detrimental to osmotolerance. Overall, our results suggest that CAD1 negatively regulates immune responses under osmotic stress, contributing to osmotolerance in Arabidopsis.

Mutations in nuclear pore complex promote osmotolerance in Arabidopsis by suppressing the nuclear translocation of ACQOS and its osmotically induced immunity.

We have previously reported a wide variation in salt tolerance among Arabidopsis thaliana accessions and identified ACQOS, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, as the causal gene responsible for the disturbance of acquired osmotolerance induced after mild salt stress. ACQOS is conserved among Arabidopsis osmosensitive accessions, including Col-0. In response to osmotic stress, it induces detrimental autoimmunity, resulting in suppression of osmotolerance, but how ACQOS triggers autoimmunity remains unclear. Here, we screened acquired osmotolerance (aot) mutants from EMS-mutagenized Col-0 seeds and isolated the aot19 mutant. In comparison with the wild type (WT), this mutant had acquired osmotolerance and decreased expression levels of pathogenesis-related genes. It had a mutation in a splicing acceptor site in NUCLEOPORIN 85 (NUP85), which encodes a component of the nuclear pore complex. A mutant with a T-DNA insertion in NUP85 acquired osmotolerance similar to aot19. The WT gene complemented the osmotolerant phenotype of aot19. We evaluated the acquired osmotolerance of five nup mutants of outer-ring NUPs and found that nup96, nup107, and aot19/nup85, but not nup43 or nup133, showed acquired osmotolerance. We examined the subcellular localization of the GFP-ACQOS protein and found that its nuclear translocation in response to osmotic stress was suppressed in aot19. We suggest that NUP85 is essential for the nuclear translocation of ACQOS, and the loss-of-function mutation of NUP85 results in acquired osmotolerance by suppressing ACQOS-induced autoimmunity in response to osmotic stress.

Golgi apparatus-localized CATION CALCIUM EXCHANGER4 promotes osmotolerance of Arabidopsis.

Calcium (Ca2+) is a major ion in living organisms, where it acts as a second messenger for various biological phenomena. The Golgi apparatus retains a higher Ca2+ concentration than the cytosol and returns cytosolic Ca2+ to basal levels after transient elevation in response to environmental stimuli such as osmotic stress. However, the Ca2+ transporters localized in the Golgi apparatus of plants have not been clarified. We previously found that a wild-type (WT) salt-tolerant Arabidopsis (Arabidopsis thaliana) accession, Bu-5, showed osmotic tolerance after salt acclimatization, whereas the Col-0 WT did not. Here, we isolated a Bu-5 background mutant gene, acquired osmotolerance-defective 6 (aod6), which reduces tolerance to osmotic, salt, and oxidative stresses, with a smaller plant size than the WT. The causal gene of the aod6 mutant encodes CATION CALCIUM EXCHANGER4 (CCX4). The aod6 mutant was more sensitive than the WT to both deficient and excessive Ca2+. In addition, aod6 accumulated higher Ca2+ than the WT in the shoots, suggesting that Ca2+ homeostasis is disturbed in aod6. CCX4 expression suppressed the Ca2+ hypersensitivity of the csg2 (calcium sensitive growth 2) yeast (Saccharomyces cerevisiae) mutant under excess CaCl2 conditions. We also found that aod6 enhanced MAP kinase 3/6 (MPK3/6)-mediated immune responses under osmotic stress. Subcellular localization analysis of mGFP-CCX4 showed GFP signals adjacent to the trans-Golgi apparatus network and co-localization with Golgi apparatus-localized markers, suggesting that CCX4 localizes in the Golgi apparatus. These results suggest that CCX4 is a Golgi apparatus-localized transporter involved in the Ca2+ response and plays important roles in osmotic tolerance, shoot Ca2+ content, and normal growth of Arabidopsis.

MOS4-associated complex contributes to proper splicing and suppression of ER stress under long-term heat stress in Arabidopsis.

Plants are often exposed not only to short-term (S-) but also to long-term (L-)heat stress over several consecutive days. A few Arabidopsis mutants defective in L-heat tolerance have been identified, but the molecular mechanisms are less understood for this tolerance than for S-heat stress tolerance. To elucidate the mechanisms of the former, we used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh3 and sloh63. The mutants were hypersensitive to L- but not to S-heat stress, and sloh63 was also hypersensitive to salt stress. We identified the causal genes, SLOH3 and SLOH63, both of which encoded splicing-related components of the MOS4-associated complex (MAC). This complex is widely conserved in eukaryotes and has been suggested to interact with spliceosomes. Both genes were induced by L-heat stress in a time-dependent manner, and some abnormal splicing events were observed in both mutants under L-heat stress. In addition, endoplasmic reticulum (ER) stress and subsequent unfolded protein response occurred in both mutants under L-heat stress and were especially prominent in sloh63, suggesting that enhanced ER stress is due to the salt hypersensitivity of sloh63. Splicing inhibitor pladienolide B led to concentration-dependent disturbance of splicing, decreased L-heat tolerance, and enhanced ER stress. These findings suggest that maintenance of precise mRNA splicing under L-heat stress by the MAC is important for L-heat tolerance and suppressing ER stress in Arabidopsis.

LHT1/MAC7 contributes to proper alternative splicing under long-term heat stress and mediates variation in the heat tolerance of Arabidopsis.

Natural genetic variation has facilitated the identification of genes underlying complex traits such as stress tolerances. We here evaluated the long-term (L-) heat tolerance (37°C for 5 days) of 174 Arabidopsis thaliana accessions and short-term (S-) heat tolerance (42°C, 50 min) of 88 accessions and found extensive variation, respectively. Interestingly, L-heat-tolerant accessions are not necessarily S-heat tolerant, suggesting that the tolerance mechanisms are different. To elucidate the mechanisms underlying the variation, we performed a chromosomal mapping using the F2 progeny of a cross between Ms-0 (a hypersensitive accession) and Col-0 (a tolerant accession) and found a single locus responsible for the difference in L-heat tolerance between them, which we named Long-term Heat Tolerance 1 (LHT1). LHT1 is identical to MAC7, which encodes a putative RNA helicase involved in mRNA splicing as a component of the MOS4 complex. We found one amino acid deletion in LHT1 of Ms-0 that causes a loss of function. Arabidopsis mutants of other core components of the MOS4 complex-mos4-2, cdc5-1, mac3a mac3b, and prl1 prl2-also showed hypersensitivity to L-heat stress, suggesting that the MOS4 complex plays an important role in L-heat stress responses. L-heat stress induced mRNA processing-related genes and compromised alternative splicing. Loss of LHT1 function caused genome-wide detrimental splicing events, which are thought to produce nonfunctional mRNAs that include retained introns under L-heat stress. These findings suggest that maintaining proper alternative splicing under L-heat stress is important in the heat tolerance of A. thaliana.

Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

Abscisic acid-mediated sugar responses are essential for vegetative desiccation tolerance in the liverwort Marchantia polymorpha.

Low-molecular-weight sugars serve as protectants for cellular membranes and macromolecules under the condition of dehydration caused by environmental stress such as desiccation and freezing. These sugars also affect plant growth and development by provoking internal signaling pathways. We previously showed that both sugars and the stress hormone abscisic acid (ABA) enhance desiccation tolerance of gemma, a dormant propagule of the liverwort Marchantia polymorpha. To determine the role of ABA in sugar responses in liverworts, we generated genome-editing lines of M. polymorpha ABA DEFICIENT 1 (MpABA1) encoding zeaxanthin epoxidase, which catalyzes the initial reaction toward ABA biosynthesis. The generated Mpaba1 lines that accumulated only a trace amount of endogenous ABA showed reduced desiccation tolerance and reduced sugar responses. RNA-seq analysis of sucrose-treated gemmalings of M. polymorpha revealed that expression of a large part of sucrose-induced genes was reduced in Mpaba1 compared to the wild-type. Furthermore, Mpaba1 accumulated smaller amounts of low-molecular-weight sugars in tissues upon sucrose treatment than the wild-type, with reduced expression of genes for sucrose synthesis, sugar transporters, and starch-catabolizing enzymes. These results indicate that endogenous ABA plays a role in the regulation of the positive feedback loop for sugar-induced sugar accumulation in liverworts, enabling the tissue to have desiccation tolerance.

Targeted in vivo mutagenesis of a sensor histidine kinase playing an essential role in ABA signaling of the moss Physcomitrium patens.

Land plants exhibit various adaptation responses to unfavorable water environments, such as drought and flooding. The phytohormone abscisic acid (ABA) and ethylene play essential roles in plant adaptation to drought and flooding, respectively. It remains largely unknown how plants integrate environmental information for water availability. In the moss Physcomitrium patens, we recently reported that not only ethylene/flooding signaling but also ABA/osmostress signaling are mediated by ethylene receptor-related sensor histidine kinases (ETR-HKs). Subfamily I ETR-HKs of this moss were found to interact with a RAF kinase (ARK) and were required for ABA-dependent activation of SNF1-related protein kinase 2 (SnRK2) via ARK activation. To elucidate the mechanisms of ARK regulation by ETR-HKs, here we employed targeted in vivo mutagenesis of PpHK5, a member of subfamily I ETR-HKs. Analyses of ABA-insensitive Pphk5 mutants indicated that PpHK5 mutations affecting the interaction with ARK resulted in loss of PpHK5 function to activate ABA signaling. We also identified a PpHK5 mutation that does not affect ARK interaction but resulted in loss of PpHK5 function. These results suggest that physical interaction between ETR-HK and ARK is essential but not sufficient for the regulation of ARK activity, and the C-terminal response regulator domain is involved in regulating ARK activation.

The seroprevalence of neutralizing antibodies against the adeno-associated virus capsids in Japanese hemophiliacs.

Adeno-associated virus (AAV) vectors are promising modalities of gene therapy to address unmet medical needs. However, anti-AAV neutralizing antibodies (NAbs) hamper the vector-mediated therapeutic effect. Therefore, NAb prevalence in the target population is vital in designing clinical trials with AAV vectors. Hence, updating the seroprevalence of anti-AAV NAbs, herein we analyzed sera from 100 healthy individuals and 216 hemophiliacs in Japan. In both groups, the overall seroprevalence against various AAV serotypes was 20%-30%, and the ratio of the NAb-positive population increased with age. The seroprevalence did not differ between healthy participants and hemophiliacs and was not biased by the concomitant blood-borne viral infections. The high neutralizing activity, which strongly inhibits the transduction with all serotypes in vitro, was mostly found in people in their 60s or of older age. The multivariate analysis suggested that "60s or older age" was the only independent factor related to the high titer of NAbs. Conversely, a large proportion of younger hemophiliacs was seronegative, rendering them eligible for AAV-mediated gene therapy in Japan. Compared with our previous study, the peak of seroprevalences has shifted to older populations, indicating that natural AAV exposure in the elderly occurred in their youth but not during the last decade.

Differential regulations of abscisic acid-induced desiccation tolerance and vegetative dormancy by group B3 Raf kinases in liverworts.

Phytohormone abscisic acid (ABA) plays a key role in stomata closure, osmostress acclimation, and vegetative and embryonic dormancy. Group B3 Raf protein kinases (B3-Rafs) serve as positive regulators of ABA and osmostress signaling in the moss Physcomitrium patens and the angiosperm Arabidopsis thaliana. While P. patens has a single B3-Raf called ARK, specific members of B3-Rafs among six paralogs regulate ABA and osmostress signaling in A. thaliana, indicating functional diversification of B3-Rafs in angiosperms. However, we found that the liverwort Marchantia polymorpha, belonging to another class of bryophytes, has three paralogs of B3-Rafs, MpARK1, MpARK2, and MpARK3, with structural variations in the regulatory domains of the polypeptides. By reporter assays of the P. patens ark line and analysis of genome-editing lines of M. polymorpha, we found that these B3-Rafs are functionally redundant in ABA response, with respect to inhibition of growth, tolerance to desiccation and expression of stress-associated transcripts, the majority of which are under the control of the PYR/PYL/RCAR-like receptor MpPYL1. Interestingly, gemmae in gemma cups were germinating only in mutant lines associated with MpARK1, indicating that dormancy in the gametophyte is controlled by a specific B3-Raf paralog. These results indicated not only conservation of the role of B3-Rafs in ABA and osmostress response in liverworts but also functional diversification of B3-Rafs, which is likely to have occurred in the early stages of land plant evolution.

KLU/CYP78A5, a Cytochrome P450 Monooxygenase Identified via Fox Hunting, Contributes to Cuticle Biosynthesis and Improves Various Abiotic Stress Tolerances.

Acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana (Arabidopsis) accessions. Most salt-tolerant accessions exhibit acquired osmotolerance, whereas Col-0 does not. To identify genes that can confer acquired osmotolerance to Col-0 plants, we performed full-length cDNA overexpression (FOX) hunting using full-length cDNAs of halophyte Eutrema salsugineum, a close relative of Arabidopsis. We identified EsCYP78A5 as a gene that can confer acquired osmotolerance to Col-0 wild-type (WT) plants. EsCYP78A5 encodes a cytochrome P450 monooxygenase and the Arabidopsis ortholog is known as KLU. We also demonstrated that transgenic Col-0 plants overexpressing AtKLU (AtKLUox) exhibited acquired osmotolerance. Interestingly, KLU overexpression improved not only acquired osmotolerance but also osmo-shock, salt-shock, oxidative, and heat-stress tolerances. Under normal conditions, the AtKLUox plants showed growth retardation with shiny green leaves. The AtKLUox plants also accumulated higher anthocyanin levels and developed denser cuticular wax than WT plants. Compared to WT plants, the AtKLUox plants accumulated significantly higher levels of cutin monomers and very-long-chain fatty acids, which play an important role in the development of cuticular wax and membrane lipids. Endoplasmic reticulum (ER) stress induced by osmotic or heat stress was reduced in AtKLUox plants compared to WT plants. These findings suggest that KLU is involved in the cuticle biosynthesis, accumulation of cuticular wax, and reduction of ER stress induced by abiotic stresses, leading to the observed abiotic stress tolerances.

ECERIFERUM 10 Encoding an Enoyl-CoA Reductase Plays a Crucial Role in Osmotolerance and Cuticular Wax Loading in Arabidopsis.

Acquired osmotolerance induced after salt stress is widespread across Arabidopsis thaliana (Arabidopsis) accessions (e.g., Bu-5). However, it remains unclear how this osmotolerance is established. Here, we isolated a mutant showing an acquired osmotolerance-defective phenotype (aod2) from an ion-beam-mutagenized M2 population of Bu-5. aod2 was impaired not only in acquired osmotolerance but also in osmo-shock, salt-shock, and long-term heat tolerances compared with Bu-5, and it displayed abnormal morphology, including small, wrinkled leaves, and zigzag-shaped stems. Genetic analyses of aod2 revealed that a 439-kbp region of chromosome 4 was translocated to chromosome 3 at the causal locus for the osmosensitive phenotype. The causal gene of the aod2 phenotype was identical to ECERIFERUM 10 (CER10), which encodes an enoyl-coenzyme A reductase that is involved in the elongation reactions of very-long-chain fatty acids (VLCFAs) for subsequent derivatization into cuticular waxes, storage lipids, and sphingolipids. The major components of the cuticular wax were accumulated in response to osmotic stress in both Bu-5 WT and aod2. However, less fatty acids, primary alcohols, and aldehydes with chain length ≥ C30 were accumulated in aod2. In addition, aod2 exhibited a dramatic reduction in the number of epicuticular wax crystals on its stems. Endoplasmic reticulum stress mediated by bZIP60 was increased in aod2 under osmotic stress. The only cer10 showed the most pronounced loss of epidermal cuticular wax and most osmosensitive phenotype among four Col-0-background cuticular wax-related mutants. Together, the present findings suggest that CER10/AOD2 plays a crucial role in Arabidopsis osmotolerance through VLCFA metabolism involved in cuticular wax formation and endocytic membrane trafficking.

MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity.

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

Mitochondrial Fission Complex Is Required for Long-Term Heat Tolerance of Arabidopsis.

Plants are often exposed not only to short-term (S) heat stress but also to long-term (L) heat stress over several consecutive days. A few Arabidopsis mutants defective in L-heat tolerance have been identified, but the molecular mechanisms involved are less well understood than those involved in S-heat tolerance. To elucidate the mechanisms, we isolated the new sensitive to long-term heat5 (sloh5) mutant from EMS-mutagenized seeds of L-heat-tolerant Col-0. The sloh5 mutant was hypersensitive to L-heat but not to S-heat, osmo-shock, salt-shock or oxidative stress. The causal gene, SLOH5, is identical to elongatedmitochondria1 (ELM1), which plays an important role in mitochondrial fission in conjunction with dynamin-related proteins DRP3A and DRP3B. Transcript levels of ELM1, DRP3A and DRP3B were time-dependently increased by L-heat stress, and drp3a drp3b double mutants were hypersensitive to L-heat stress. The sloh5 mutant contained massively elongated mitochondria. L-heat stress caused mitochondrial dysfunction and cell death in sloh5. Furthermore, WT plants treated with a mitochondrial myosin ATPase inhibitor were hypersensitive to L-heat stress. These findings suggest that mitochondrial fission and function are important in L-heat tolerance of Arabidopsis.

Sensor histidine kinases mediate ABA and osmostress signaling in the moss Physcomitrium patens.

To survive fluctuating water availability on land, terrestrial plants must be able to sense water stresses, such as drought and flooding. The plant hormone abscisic acid (ABA) and plant-specific SNF1-related protein kinase 2 (SnRK2) play key roles in plant osmostress responses. We recently reported that, in the moss Physcomitrium patens, ABA and osmostress-dependent SnRK2 activation requires phosphorylation by an upstream RAF-like kinase (ARK). This RAF/SnRK2 module is an evolutionarily conserved mechanism of osmostress signaling in land plants. Surprisingly, ARK is also an ortholog of Arabidopsis CONSTITUTIVE RESPONSE 1 (CTR1), which negatively regulates the ethylene-mediated submergence response of P. patens, indicating a nexus for cross-talk between the two signaling pathways that regulate responses to water availability. However, the mechanism through which the ARK/SnRK2 module is activated in response to water stress remains to be elucidated. Here, we show that a group of ethylene-receptor-related sensor histidine kinases (ETR-HKs) is essential for ABA and osmostress responses in P. patens. The intracellular kinase domain of an ETR-HK from P. patens physically interacts with ARK at the endoplasmic reticulum in planta. Moreover, HK disruptants lack ABA-dependent autophosphorylation of the critical serine residue in the activation loop of ARK, leading to loss of SnRK2 activation in response to ABA and osmostress. Collectively with the notion that ETR-HKs participate in submergence responses, our present data suggest that the HK/ARK module functions as an integration unit for environmental water availability to elicit optimized water stress responses in the moss P. patens.

Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.

Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin- and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin- in liver sinusoidal endothelial cells.

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses.

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

An ER-Golgi Tethering Factor SLOH4/MIP3 Is Involved in Long-Term Heat Tolerance of Arabidopsis.

Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L-heat stress but not to S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2 and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1-1 mutants was similar to that of the wild type (WT). Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the WT. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1) - accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins - were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not by bZIP28, resulting in the initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in the MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER-Golgi vesicle tethering.

CATALASE2 plays a crucial role in long-term heat tolerance of Arabidopsis thaliana.

Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days; nevertheless, most previous studies of heat tolerance have used S-heat stress, such as 42 °C for 30-60 min, for evaluation. Yet the mechanisms underlying L-heat tolerance remain poorly understood. Here we found that hydrogen peroxide (H2O2) in Arabidopsis thaliana plants increased time-dependently under L-heat stress (37 °C, 5 days) but not under S-heat stress (42 °C, 40 min). To reveal the contribution of reactive oxygen species (ROS) scavenging to heat tolerance, we evaluated the heat tolerance of ROS mutants. Only cat2 mutants, in which catalase (CAT) activity is defective, were hypersensitive to L-heat stress, but they were S-heat tolerant. We further revealed that (1) CAT2 was induced by L-heat stress but not by S-heat stress; (2) H2O2 accumulated highly in cat2 under L-heat stress, but not in cat1, cat3, or wild type; and (3) CAT activity was significantly reduced in cat2 under both normal and L-heat conditions. These results suggest that ROS scavenging is responsible for L-heat tolerance, and CAT2 plays a crucial role. On the other hand, since overexpression of CAT2 in wild-type plants did not enhance L-heat tolerance, CAT2 activity is necessary but insufficient for increasing L-heat tolerance.