Water availability is a key determinant of terrestrial plant productivity. Many climate models predict that water stress will increasingly challenge agricultural yields and exacerbate projected food ...deficits. To ensure food security and increase agricultural efficiency, crop water productivity must be increased. Research over past decades has established that the phytohormone abscisic acid (ABA) is a central regulator of water use and directly regulates stomatal opening and transpiration. In this study, we investigated whether the water productivity of wheat could be improved by increasing its ABA sensitivity. We show that overexpression of a wheat ABA receptor increases wheat ABA sensitivity, which significantly lowers a plant's lifetime water consumption. Physiological analyses demonstrated that this water-saving trait is a consequence of reduced transpiration and a concomitant increase in photosynthetic activity, which together boost grain production per litre of water and protect productivity during water deficit. Our findings provide a general strategy for increasing water productivity that should be applicable to other crops because of the high conservation of the ABA signalling pathway.
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.
•CAT2 plays an important role in long-term, but not short-term, heat tolerance.•CAT2 expression is increased under long-term, but not short-term, heat stress.•H2O2 and cell death are induced in cat2 mutant under long-term heat stress.•Overexpression of CAT2 did not enhance long-term heat tolerance.
SUMMARY
Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 ...(SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.
Significance Statement
Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) belonging to the B3 clade of the mitogen‐activated kinase kinase kinase family play a crucial role in Arabidopsis drought responses, such as stomatal closure, through phosphorylation‐mediated activation of subclass III SNF1‐related protein kinase 2 (SnRK2) partly in an abscisic acid‐independent manner. Our findings indicate that ARK is a novel component of osmostress signaling that connects osmosensing with SnRK2 activation.
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment ...strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
Abstract
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 CD31
high
, CD146
high
, 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 CD31
high
, CD146
high
, Lyve1
+
, CLEC-2
+
, and podoplanin
−
in liver sinusoidal endothelial cells.
Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or ...disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis.
A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α2-plasmin inhibitor (PI), plasmin-α2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days.
A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three together, significantly improved the AUROC up to 0.95 (vs. TAT, PAI-1, and PC). Among the significant diagnostic markers for overt DIC, TAT and PAI-1 were also good predictors of 28-day mortality (AUROC, 0.77 and 0.81, respectively).
Severe coagulation and fibrinolytic abnormalities on ICU admission were associated with subsequent development of overt DIC. A single measurement of TAT, PAI-1, and PC activity could identify patients with ongoing severe coagulopathy, early in the course of sepsis.
Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as ...hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the mossPhyscomitrella patenswith reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene inP. patensbelongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.
Dormancy is a key process allowing land plants to adapt to changing conditions in the terrestrial habitat, allowing the cessation of growth in response to environmental or physiological cues, ...entrance into a temporary quiescent state, and subsequent reactivation of growth in more favorable environmental conditions 1–3. Dormancy may be induced seasonally, sporadically (e.g., in response to drought), or developmentally (e.g., seeds and apical dominance). Asexual propagules, known as gemmae, derived via clonal reproduction in bryophytes, are often dormant until displaced from the parent plant. In the liverwort Marchantia polymorpha, gemmae are produced within specialized receptacles, gemma cups, located on the dorsal side of the vegetative thallus 4. Mature gemmae are detached from the parent plant but may remain in the cup, with gemma growth suppressed as long as the gemmae remain in the gemma cup and the parental plant is alive 5. Following dispersal of gemmae from gemma cups by rain, the gemmae germinate in the presence of light and moisture, producing clonal offspring 6. In land plants, the plant hormone abscisic acid (ABA) regulates many aspects of dormancy and water balance 7. Here, we demonstrate that ABA plays a central role in the control of gemma dormancy as transgenic M. polymorpha gemmae with reduced sensitivity to ABA fail to establish and/or maintain dormancy. Thus, the common ancestor of land plants used the ABA signaling module to regulate germination of progeny in response to environmental cues, with both gemmae and seeds being derived structures co-opting an ancestral response system.
•ABA delays Marchantia polymorpha gemma germination in a dose-dependent manner•ABA-insensitive ABI1 gain-of-function alleles display reduced gemma dormancy•ABA-insensitive abi3 loss-of-function alleles display reduced gemma dormancy•ABA signaling acts in the gemmae themselves to promote dormancy
Eklund et al. show that in the liverwort Marchantia polymorpha, abscisic acid promotes dormancy in vegetative propagules known as gemmae. Their results indicate that an ABA signaling pathway, including orthologs of ABI1 and ABI3, operates locally in the gemmae themselves and likely acts in response to a long-range dormancy signal.
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.
Abstract
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.
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