A conventional fixation volume electron microscopy protocol for plants.

Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.

Redox responses of Arabidopsis thaliana to the green peach aphid, Myzus persicae.

The green peach aphid (Myzus persicae) is a phloem-feeding insect that causes economic damage on a wide array of crops. Using a luminol-based assay, a superoxide-responsive reporter gene (Zat12::luciferase), and a probe specific to hydrogen peroxide (HyPer), we demonstrated that this aphid induces accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. Similar to the apoplastic oxidative burst induced by pathogens, this response to aphids was rapid and transient, with two peaks occurring within 1 and 4 hr after infestation. Aphid infestation also induced an oxidative response in the cytosol and peroxisomes, as measured using a redox-sensitive variant of green fluorescent protein (roGFP2). This intracellular response began within minutes of infestation but persisted 20 hr or more after inoculation, and the response of the peroxisomes appeared stronger than the response in the cytosol. Our results suggest that the oxidative response to aphids involves both apoplastic and intracellular sources of ROS, including ROS generation in the peroxisomes, and these different sources of ROS may potentially differ in their impacts on host suitability for aphids.

Loss of Function of Fatty Acid Desaturase 7 in Tomato Enhances Photosynthetic Carbon Fixation Efficiency.

Fatty Acid Desaturase 7 (FAD7) generates polyunsaturated fatty acids, promoting the desaturation of chloroplast membranes; it also provides an essential precursor for the synthesis of jasmonic acid (JA), a phytohormone that can influence plant growth, development, and primary metabolism. This study examined the effects of spr2, a null mutation in SlFAD7, on the growth, morphology, and photosynthetic traits of tomato, Solanum lycopersicum. Although the spr2 mutant had a lower density of stomata than wild type plants, the two genotypes had comparable stomatal conductance, transpiration rates, and intracellular CO2 levels; in addition, spr2 had significantly thinner leaf blades, which may help maintain normal levels of CO2 diffusion despite the lower number of stomata. Surprisingly, spr2 also had significantly higher carbon assimilation (A) and maximum quantum efficiency of PSII (Fv/Fm) than wild type plants at both of the light intensities tested here (220 or 440 µmol m-2 s-1), despite having lower levels of chlorophyll than wild type plants under low light (220 µmol m-2 s-1). Furthermore, CO2 response curves indicated higher in vivo Rubisco activity (Vcmax) in spr2 compared to wild type plants, as well as an enhanced maximum rate of electron transport used in the regeneration of ribulose-1,5-bisphosphate (Jmax). These data indicate that loss of function of FAD7 can enhance the efficiency of both light-dependent and light-independent reactions in photosynthesis. Consistent with this, the spr2 mutant also displayed enhanced growth, with significantly more leaves and a more compact growth habit. In contrast to spr2, another tomato mutant impaired in JA synthesis (acx1) showed no enhancements in growth or photosynthetic efficiency, suggesting that the enhancements observed in spr2 are independent of the effects of this mutation on JA synthesis. These data demonstrate that loss of function of FAD7 can enhance photosynthesis and growth, potentially through its impacts on the chloroplast membranes.