Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

Dynamic calcium signals mediate the feeding response of the carnivorous sundew plant.

Some of the most spectacular examples of botanical carnivory-in which predator plants catch and digest animals presumably to supplement the nutrient-poor soils in which they grow-occur within the Droseraceae family. For example, sundews of the genus Drosera have evolved leaf movements and enzyme secretion to facilitate prey digestion. The molecular underpinnings of this behavior remain largely unknown; however, evidence suggests that prey-induced electrical impulses are correlated with movement and production of the defense hormone jasmonic acid (JA), which may alter gene expression. In noncarnivorous plants, JA is linked to electrical activity via changes in cytoplasmic Ca2+. Here, we find that dynamic Ca2+ changes also occur in sundew (Drosera spatulata) leaves responding to prey-associated mechanical and chemical stimuli. Furthermore, inhibition of these Ca2+ changes reduced expression of JA target genes and leaf movements following chemical feeding. Our results are consistent with the presence of a conserved Ca2+-dependent JA signaling pathway in the sundew feeding response and provide further credence to the defensive origin of plant carnivory.

Moss PIEZO homologs have a conserved structure, are ubiquitously expressed, and do not affect general vacuole function.

The PIEZO protein family was first described in animals where these mechanosensitive calcium channels perform numerous essential functions, including the perception of light touch, shear, and compressive forces. PIEZO homologs are present in most eukaryotic lineages and recently we reported that two PIEZO homologs from moss Physcomitrium patens localize to the vacuolar membrane and modulate its morphology in tip-growing caulonemal cells. Here we show that predicted structures of both PpPIEZO1 and PpPIEZO2 are very similar to that of mouse Piezo2. Furthermore, we show that both moss PIEZO genes are ubiquitously expressed in moss vegetative tissues and that they are not required for normal vacuolar pH or intracellular osmotic potential. These results suggest that moss PIEZO proteins are widely expressed mechanosensory calcium channels that serve a signaling rather than maintenance role in vacuoles.

Plant PIEZO homologs modulate vacuole morphology during tip growth.

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana PpPIEZO1 and PpPIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both PpPIEZO1 and PpPIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.

Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL1, MSL2, and MSL3 reveal a role for inter-organellar communication in plant development.

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between three Mechanosensitive channel of Small conductance-Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bacterial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to the inner mitochondrial membrane, while MSL2 and MSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion in MSL1, both in wild type and in msl2 msl3 mutant backgrounds. msl1 single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling in msl2 msl3 double mutants was exacerbated in the msl1 msl2 msl3 triple mutant. However, the introduction of the msl1 lesion into the msl2 msl3 mutant background suppressed other msl2 msl3 mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version of MSL1. In yeast-based interaction studies, MSL1 interacted with itself, but not with MSL2 or MSL3. These results establish that the abnormalities observed in msl2 msl3 double mutants is partially dependent on the presence of functional MSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

Heritable Phytohormone Profiles of Poplar Genotypes Vary in Resistance to a Galling Aphid.

Insect galls are highly specialized structures arising from atypical development of plant tissue induced by insects. Galls provide the insect enhanced nutrition and protection against natural enemies and environmental stresses. Galls are essentially plant organs formed by an intimate biochemical interaction between the gall-inducing insect and its host plant. Because galls are plant organs, their development is likely to be governed by phytohormones involved in normal organogenesis. We characterized concentrations of both growth and defensive phytohormones in ungalled control leaves and galls induced by the aphid Pemphigus betae on narrowleaf cottonwood Populus angustifolia that differ genotypically in resistance to this insect. We found that susceptible trees differed from resistant trees in constitutive concentrations of both growth and defense phytohormones. Susceptible trees were characterized by significantly higher constitutive cytokinin concentrations in leaves, significantly greater ability of aphids to elicit cytokinin increases, and significantly lower constitutive defense phytohormone concentrations than observed in resistant trees. Phytohormone concentrations in both constitutive and induced responses in galled leaves exhibited high broad-sense heritability that, respectively, ranged from 0.39 to 0.93 and from 0.28 to 0.66, suggesting that selection can act upon these traits and that they might vary across the landscape. Increased cytokinin concentrations may facilitate forming strong photosynthate sinks in the galls, a requirement for galling insect success. By characterizing for the first time the changes in 15 phytohormones belonging to five different classes, this study offers a better overview of the signaling alteration occurring in galls that has likely been important for their ecology and evolution. Copyright © 2019 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

Morphometric analysis of young petiole galls on the narrow-leaf cottonwood, Populus angustifolia, by the sugarbeet root aphid, Pemphigus betae.

An insect-induced gall is a highly specialized structure resulting from atypical development of plant tissue induced by a reaction to the presence and activity of an insect. The insect induces a differentiation of tissues with features and functions of an ectopic organ, providing nutrition and protection to the galling insect from natural enemies and environmental stresses. In this anatomical and cytological study, we characterized how the gall-inducing aphid Pemphigus betae reshapes the leaf morphology of the narrow-leaf cottonwood Populus angustifolia to form a leaf fold gall. Young galls displayed a bend on one side of the midvein toward the center of the leaf and back to create a fold on the abaxial side of the leaf. This fold was formed abaxially by periclinal and anticlinal divisions, effectively eliminating intercellular spaces from the spongy parenchyma. Galls at this stage exhibited both cell hypertrophy and tissue hyperplasia. Cells on the adaxial surface were more numerous and smaller than cells near the abaxial surface were, creating the large fold that surrounds the insect. Mesophyll cells exhibited some features typical of nutritive cells induced by other galling insects, including conspicuous nucleolus, reduced and fragmented vacuole, smaller and degraded chloroplasts, and dense cytoplasm compared to ungalled tissue. Even though aphids feed on the contents of phloem and do not directly consume the gall tissue, they induce changes in the plant vascular system, which lead to nutrient accumulation to support the growing aphid numbers in mature galls.