A missense mutation in CHS1, a TIR‐NB protein,induces chilling sensitivity in Arabidopsis

Low temperature is an environmental factor that affects plant growth and development and plant–pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling‐sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling‐sensitive phenotype, and also displayed defense‐associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map‐based cloning analysis revealed that CHS1 encodes a TIR‐NB‐type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1–conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR‐NB‐type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR‐NB protein in Arabidopsis.     

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28 °C than at 22 °C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.     

Can chilling tolerance of C4 photosynthesis in Miscanthus be transferred to sugarcane?

The goal of this study was to investigate whether chilling tolerance of C₄ photosynthesis in Miscanthus can be transferred to sugarcane by hybridization. Net leaf CO₂ uptake (A_sat) and the maximum operating efficiency of photosystem II (Ф_PSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 day in controlled environment chambers. Two of three hybrids (miscanes), ‘US 84‐1058’ and ‘US 87‐1019’, did not differ significantly from the chilling tolerant M. ×giganteus ‘Illinois’ (Mxg), for A_sat, and Φ_PSII measured during chilling. For Mxg grown at 10 °C/5 °C for 11 days, A_sat was 4.4 μmol m^?2 s^?1, while for miscane ‘US 84‐1058’ and ‘US 87‐1019’, A_sat was 5.7 and 3.5 μmol m^?2 s^?1, respectively. Miscanes ‘US 84‐1058’ and ‘US 87‐1019’ and Mxg had significantly higher rates of A_sat during chilling than three tested sugarcanes. A third miscane showed lower rates than Mxg during chilling, but recovered to higher rates than sugarcane upon return to warm conditions. Chilling tolerance of ‘US 84‐1058’ was further confirmed under autumn field conditions in southern Illinois. The selected chilling tolerant miscanes have particular value for biomass feedstock and biofuel production and at the same time they can be a starting point for extending sugarcane's range to colder climates.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

An Arabidopsis ATPase gene involved in nematode‐induced syncytium development and abiotic stress responses

The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up‐regulated in syncytia as shown by RT‐PCR, quantitative RT‐PCR, in situ RT‐PCR and promoter::GUS lines, encodes an AAA+‐type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T‐DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T‐DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the ‘meiotic clade’ of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1.     

The role of Pro-P5C Cycle in chs mutants of Arabidopsis under cold stress

Proline metabolism is implicated in plant responses to abiotic stresses, including the chilling stress. During proline catabolism, the two-step oxidation of proline is performed by the continuous actions of proline dehydrogenase (ProDH), which produces Δ¹-pyrroline-5-carboxylate (P5C), and P5C dehydrogenase (P5CDH), which oxidizes P5C to glutamate. The Arabidopsis thaliana chilling mutants chs1 and chs2 are sensitive to chilling temperatures of 13–18°C. For a better understanding of Arabidopsis responses to chilling stress, 4-week-old wild-type (WT) and chs1 and chs2 lines, with three plants in each group, were subjected to chilling stress (13°C), cold stress (4°C), or remained under normal conditions (23°C); and several factors including the expression of ProDH2 and P5CDH genes, POX (peroxidase) and SOD (superoxide dismutase) activities, as well as MDA and proline contents were examined. Our results showed an increase in the proline content in all lines under chilling conditions. In addition, a greater expression of ProDH2 and a lower expression of P5CDH were observed, leading us to speculate a greater breakdown of proline into P5C and a consequent overproduction of ROS in the ETC cycle. The higher POX and SOD activities and a higher MDA content in chs mutants at 13°C are in line with this speculation. Finally, cold-treated plants (4°C) only showed an increase in proline levels.     

Siberian Miscanthus sacchariflorus accessions surpass the exceptional chilling tolerance of the most widely cultivated clone of Miscanthus x giganteus

Chilling temperatures (0–15°C) inhibit photosynthesis in most C₄ grasses, yet photosynthesis is chilling tolerant in the ‘Illinois’ clone of the C₄ grass Miscanthus x giganteus, a candidate cellulosic bioenergy crop. M. x giganteus is a hybrid between Miscanthus sacchariflorus and Miscanthus sinensis; therefore chilling‐tolerant parent lines might produce hybrids superior to the current clone. Recently a collection of M. sacchariflorus from Siberia, the apparent low temperature limit of natural distribution, became available, which may be a source for chilling tolerance. The collection was screened for chilling tolerance of photosynthesis by measuring dark‐adapted maximum quantum yield of PSII photochemistry (F_v/F_m) on plants in the field in cool weather. Superior accessions were selected for further phenotyping: plants were grown at 25°C, transferred to 10°C (chilling) for 15 days, and returned to 25°C for 7 days (recovery). Two experiments assessed: (a) light‐saturated net photosynthetic rate (A_sat) and operating quantum yield of PSII photochemistry (Φ_PSII), (b) response of net leaf CO₂ uptake (A) to intercellular [CO₂] (c_i). Three accessions showed superior chilling tolerance: RU2012‐069 and RU2012‐114 achieved A_sat up to double that of M. x giganteus prior to and during chilling, due to increased c_i ‐ saturated photosynthesis (V_max). RU2012‐069 and RU2012‐114 also maintained greater levels of Φ_PSII during chilling, indicating reduced photodamage. Additionally, accession RU2012‐112 maintained a stable A_sat throughout the 15‐day chilling period, while A_sat continuously declined in other accessions; this suggests RU2012‐112 could outperform others in lengthy chilling periods. Plants were returned to 25°C after the chilling period; M. x giganteus showed the weakest recovery after 1 day, but a strong recovery after 1 week. This study has therefore identified important genetic resources for the synthesis of improved lines of M. x giganteus, which could facilitate the displacement of fossil fuels by cellulosic bioenergy.     

IBR5 Modulates Temperature-Dependent,R Protein CHS3-Mediated Defense Responses in Arabidopsis

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.     

DOWNY MILDEW RESISTANT 6 and DMR6‐LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis

Arabidopsis downy mildew resistant 6 (dmr6) mutants have lost their susceptibility to the downy mildew Hyaloperonospora arabidopsidis. Here we show that dmr6 is also resistant to the bacterium Pseudomonas syringae and the oomycete Phytophthora capsici. Resistance is accompanied by enhanced defense gene expression and elevated salicylic acid levels. The suppressive effect of the DMR6 oxygenase was confirmed in transgenic Arabidopsis lines overexpressing DMR6 that show enhanced susceptibility to H. arabidopsidis, P. capsici, and P. syringae. Phylogenetic analysis of the superfamily of 2‐oxoglutarate Fe(II)‐dependent oxygenases revealed a subgroup of DMR6‐LIKE OXYGENASEs (DLOs). Within Arabidopsis, DMR6 is most closely related to DLO1 and DLO2. Overexpression of DLO1 and DLO2 in the dmr6 mutant restored the susceptibility to downy mildew indicating that DLOs negatively affect defense, similar to DMR6. DLO1, but not DLO2, is co‐expressed with DMR6, showing strong activation during pathogen attack and following salicylic acid treatment. DMR6 and DLO1 differ in their spatial expression pattern in downy mildew‐infected Arabidopsis leaves; DMR6 is mostly expressed in cells that are in contact with hyphae and haustoria of H. arabidopsidis, while DLO1 is expressed mainly in the vascular tissues near infection sites. Strikingly, the dmr6‐3_dlo1 double mutant, that is completely resistant to H. arabidopsidis, showed a strong growth reduction that was associated with high levels of salicylic acid. We conclude that DMR6 and DLO1 redundantly suppress plant immunity, but also have distinct activities based on their differential localization of expression.     

Rapid thermal adaptation in photosymbionts of reef‐building corals

Climate warming is occurring at a rate not experienced by life on Earth for 10 s of millions of years, and it is unknown whether the coral‐dinoflagellate (Symbiodinium spp.) symbiosis can evolve fast enough to ensure coral reef persistence. Coral thermal tolerance is partly dependent on the Symbiodinium hosted. Therefore, directed laboratory evolution in Symbiodinium has been proposed as a strategy to enhance coral holobiont thermal tolerance. Using a reciprocal transplant design, we show that the upper temperature tolerance and temperature tolerance range of Symbiodinium C1 increased after ~80 asexual generations (2.5 years) of laboratory thermal selection. Relative to wild‐type cells, selected cells showed superior photophysiological performance and growth rate at 31°C in vitro, and performed no worse at 27°C; they also had lower levels of extracellular reactive oxygen species (exROS). In contrast, wild‐type cells were unable to photosynthesise or grow at 31°C and produced up to 17 times more exROS. In symbiosis, the increased thermal tolerance acquired ex hospite was less apparent. In recruits of two of three species tested, those harbouring selected cells showed no difference in growth between the 27 and 31°C treatments, and a trend of positive growth at both temperatures. Recruits that were inoculated with wild‐type cells, however, showed a significant difference in growth rates between the 27 and 31°C treatments, with a negative growth trend at 31°C. There were no significant differences in the rate and severity of bleaching in coral recruits harbouring wild‐type or selected cells. Our findings highlight the need for additional Symbiodinium genotypes to be tested with this assisted evolution approach. Deciphering the genetic basis of enhanced thermal tolerance in Symbiodinium and the cause behind its limited transference to the coral holobiont in this genotype of Symbiodinium C1 are important next steps for developing methods that aim to increase coral bleaching tolerance.     

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.