Arabidopsis thaliana Cuticle Composition Contributes to Differential Defense Response to Botrytis cinerea

The chemical composition of a plant cuticle can change in response to various abiotic or biotic stresses and plays essential functions in disease resistance responses. Arabidopsis thaliana mutants altered in cutin content are resistant to Botrytis cinerea, presumably because of increased cuticular water and solute permeability, allowing for faster induction of defense responses. Within this context, our knowledge of wax mutants is limited against this pathogen. We tested the contribution of cuticular components to immunity to B. cinerea using mutants altered in either cutin or wax alone, or in both cutin and wax contents. We found that even all the tested mutants showed increased permeability and reactive oxygen species (ROS) accumulation in comparison with wild-type plants and that only cutin mutants showed resistance. To elucidate the early molecular mechanisms underlying cuticle-related immunity, we performed a transcriptomic analysis. A set of upregulated genes involved in cell wall integrity and accumulation of ROS were shared by the cutin mutants bdg, lacs2-3, and eca2, but not by the wax mutants cer1-4 and cer3-6. Interestingly, these genes have recently been shown to be required in B. cinerea resistance. In contrast, we found the induction of genes involved in abiotic stress shared by the two wax mutants. Our study reveals new insight that the faster recognition of a pathogen by changes in cuticular permeability is not enough to induce resistance to B. cinerea, as has previously been hypothesized. In addition, our data suggest that mutants with resistant phenotype can activate other defense pathways, different from those canonical immune ones.

A modified method for enzymatic isolation of and subsequent wax extraction from Arabidopsis thaliana leaf cuticle

Background The plant cuticle represents one of the major adaptations of vascular plants to terrestrial life. Cuticular permeability and chemical composition differ among species. Arabidopsis thaliana is a widely used model for biochemical and molecular genetic studies in plants. However, attempts to isolate the intact cuticle from fresh leaves of Arabidopsis have failed so far. The goal of this study was to optimise an enzymatic method for cuticle isolation of species with a thin cuticle and to test it on several A. thaliana wild types and mutants. Results We developed a method for isolation of thin cuticles that allows reducing the isolation time, the separation of abaxial and adaxial cuticles, and avoids formation of wrinkles. Optical microscopy was used for studying cuticle intactness and scanning electron microscopy for visualisation of external and internal cuticle structures after isolation. Wax extracts were analysed by GC–MS. Isolation of intact cuticle was successful for all tested plants. The wax compositions (very-long-chained fatty acids, alcohols and alkanes) of intact leaves and isolated cuticles of wild type Col-0 were compared. Conclusions We conclude that the optimised enzymatic method is suitable for the isolation of A. thaliana adaxial and abaxial cuticles. The isolated cuticles are suitable for microscopic observation. Analysis of wax composition revealed some discrepancies between isolated cuticles and intact leaves with a higher yield of wax in isolated cuticles.

Comparative Cutaneous Water Loss and Desiccation Tolerance of Four Solenopsis spp. (Hymenoptera: Formicidae) in the Southeastern United States

The high surface area to volume ratio of terrestrial insects makes them highly susceptible to desiccation mainly through the cuticle. Cuticular permeability (CP) is usually the most important factor limiting water loss in terrestrial insects. Water loss rate, percentage of total body water (%TBW) content, CP, and desiccation tolerance were investigated in workers of four Solenopsis species in the southeastern USA. We hypothesized that tropical/subtropical ants (S. invicta and S. geminata) will have lower CP values and tolerate higher levels of desiccation than temperate ants (S. richteri and S. invicta × S. richteri). The %TBW content was similar among species. Solenopsis invicta had a 1.3-fold and 1.1-fold lower CP value than S. invicta × S. richteri and S. richteri, respectively. Solenopsis geminata had a 1.3-fold lower CP value than S. invicta × S. richteri, and a 1.2-fold lower CP value than S. richteri. The LT50 values (lethal time to kill 50% of the population) ranged from 1.5 h (small S. geminata) to 8.5 h (large S. invicta). Desiccation tolerance ranged between 36 and 50 %TBW lost at death and was not related to a species’ location of origin. This study is the first report of water relations of S. invicta × S. richteri. It demonstrates that desiccation stress differentially can affect the survival of different Solenopsis species and implies that environmental stress can affect the distribution of these species in the southeastern USA.

The SUPPRESSOR of MAX2 1 (SMAX1)-Like SMXL6, SMXL7 and SMXL8 Act as Negative Regulators in Response to Drought Stress in Arabidopsis

Drought represents a major threat to crop growth and yields. Strigolactones (SLs) contribute to regulating shoot branching by targeting the SUPPRESSOR OF MORE AXILLARY GROWTH2 (MAX2)-LIKE6 (SMXL6), SMXL7 and SMXL8 for degradation in a MAX2-dependent manner in Arabidopsis. Although SLs are implicated in plant drought response, the functions of the SMXL6, 7 and 8 in the SL-regulated plant response to drought stress have remained unclear. Here, we performed transcriptomic, physiological and biochemical analyses of smxl6, 7, 8 and max2 plants to understand the basis for SMXL6/7/8-regulated drought response. We found that three D53 (DWARF53)-Like SMXL members, SMXL6, 7 and 8, are involved in drought response as the smxl6smxl7smxl8 triple mutants showed markedly enhanced drought tolerance compared to wild type (WT). The smxl6smxl7smxl8 plants exhibited decreased leaf stomatal index, cuticular permeability and water loss, and increased anthocyanin biosynthesis during dehydration. Moreover, smxl6smxl7smxl8 were hypersensitive to ABA-induced stomatal closure and ABA responsiveness during and after germination. In addition, RNA-sequencing analysis of the leaves of the D53-like smxl mutants, SL-response max2 mutant and WT plants under normal and dehydration conditions revealed an SMXL6/7/8-mediated network controlling plant adaptation to drought stress via many stress- and/or ABA-responsive and SL-related genes. These data further provide evidence for crosstalk between ABA- and SL-dependent signaling pathways in regulating plant responses to drought. Our results demonstrate that SMXL6, 7 and 8 are vital components of SL signaling and are negatively involved in drought responses, suggesting that genetic manipulation of SMXL6/7/8-dependent SL signaling may provide novel ways to improve drought resistance.

Water Loss and Desiccation Tolerance of the Two Yearly Generations of Adult and Nymphal Kudzu Bugs, Megacopta cribraria (Hemiptera: Plataspidae)

Water loss rate, percentage total body water content (%TBW), cuticular permeability (CP), and desiccation tolerance were investigated in adult and immature stages of the invasive kudzu bug, Megacopta cribraria (Fab.) (Hemiptera: Plataspidae), a serious soybean pest and an urban nuisance. Adults and all five nymphal instars were weighed prior to and 2, 4, 6, 8, 10, and 24 h after desiccated at 30 ± 1°C and 0–2% RH. Both % initial mass and %TBW loss increased linearly with time of desiccation. Rates of loss ranged from approximately 1–7%/h. Mortality occurred at 10 h after desiccation. Desiccation tolerance (%TBW lost at death) ranged between 25.6% for first-generation adult females and 75% for first-generation fifth-instar nymphs. First-generation first-instar nymphs had significantly greater %TBW (88.9%) than the other generations and instars, whereas second-generation fifth instars had the lowest %TBW (62.4%). The CP value of first-generation adult females (12.3 ± 1.6 µg cm−1 h−1 mmHg−1) was the greatest across generations. First-generation first instars had the greatest mass loss (111.11 mg/g) among all instars and generations, whereas overwintered second-generation adult females had the lowest mass loss (18.39) across generations. This study demonstrated that desiccation stress differentially affected the survival of adult and nymphal kudzu bugs and may imply that environmental stress can affect the relative abundance of this species in the fields and around homes.

Cuticular Permeability, Percent Body Water Loss, and Relative Humidity Equilibria Comparisons of Four Termite Species

This study compared percentage of total body water (%TBW), water loss rate under desiccative conditions, and cuticular permeability (CP) of four termite species (Order: Blattodea, Infraorder: Isoptera) from different habitats, including one subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), one dampwood termite, Neotermes jouteli (Banks) (Blattodea: Kalotermitidae), one ‘wetwood’ termite, Cryptotermes cavifrons Banks (Blattodea: Kalotermitidae), and one drywood termite, Cryptotermes brevis Walker (Blattodea: Kalotermitidae). There was no consistent pattern associated with the habitat relative humidity (RH) levels and the %TBW among the four termite species tested. Because C. formosanus forage for food, its lowest %TBW may be due to its ability to access water sources, and its need to be more mobile than kalotermitids that remain in wood. Body water loss rate and CP were lower for xeric species such as Cr. brevis than hydric species such as N. jouteli. An RH equilibrium represents the capacity of termite water retention mechanisms that may include the physical structure of cuticular layers, behavioral manipulation of natural openings, and physiological means. As with water loss rate and CP data, RH equilibria were lower for xeric species such as Cr. brevis than hydric species such as N. jouteli. Moreover, RH equilibria for dead individuals of the four termite species were significantly higher than those of live individuals, indicating dead termites lost more water after losing their physiological and behavioral means of retaining water. RH equilibria represent termites’ capability in water retention, and there is a strong linear relationship between RH equilibria and CP values. Hence, RH equilibria may offer simpler alternatives to CP estimates.

Adult Paederus fuscipes (Coleoptera: Staphylinidae) Beetles Overcome Water Loss With Increased Total Body Water Content, Energy Metabolite Storage, and Reduced Cuticular Permeability: Age, Sex-Specific, and Mating Status Effects on Desiccation

The ability of Paederus beetles to resist desiccation stress is vital to their adaptability in various ecological niches. How water relations and their response to desiccation vary among adult beetles of different age, sex, and mating status is unclear. We examined the water relations of adult Paederus fuscipes Curtis and the mechanisms used to reduce desiccation stress. One-day-old beetles had an exceptionally high percent total body water (%TBW) content and tolerated a high level of %TBW loss. Newly emerged beetles contained a high level of trehalose and 40 to 60% lipid content of their total dry mass, which allowed them to endure desiccation. Beetles that were 10 wk old and older exhibited reduced cuticular permeability. Glucose, glycogen, and lipid contents were crucial throughout most of the adult life span, as they helped compensate for water loss via increased water vapor absorption and metabolic water. In particular, the accumulation of lipid after mating was significant and may further confer tolerance to water loss. The effect of melanization on the desiccation tolerance of beetles was not significant. Females had better tolerance in response to desiccation stress compared with males. We suggest that the observed differences between sexes likely were a function of water relations and an effect of energy metabolite reserves. However, the mortality of females at 24-h postdesiccating stage was marginally significant compared with males. These results demonstrate that P. fuscipes adults prevent dehydration using multiple mechanisms that collectively reduce desiccation stress and increase dehydration tolerance.