Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome.

Volume electron microscopy (EM) is a time-consuming process - often requiring weeks or months of continuous acquisition for large samples. In order to compare the ultrastructure of a number of individuals or conditions, acquisition times must therefore be reduced. For resin-embedded samples, one solution is to selectively target smaller regions of interest by trimming with an ultramicrotome. This is a difficult and labour-intensive process, requiring manual positioning of the diamond knife and sample, and much time and training to master. Here, we have developed a semi-automated workflow for targeting with a modified ultramicrotome. We adapted two recent commercial systems to add motors for each rotational axis (and also each translational axis for one system), allowing precise and automated movement. We also developed a user-friendly software to convert X-ray images of resin-embedded samples into angles and cutting depths for the ultramicrotome. This is provided as an open-source Fiji plugin called Crosshair. This workflow is demonstrated by targeting regions of interest in a series of Platynereis dumerilii samples.

A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis.

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling 'Structure Establishment Phase' followed by a 'Chloroplast Proliferation Phase' during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.

Mutation of the Atypical Kinase ABC1K3 Partially Rescues the PROTON GRADIENT REGULATION 6 Phenotype in Arabidopsis thaliana.

Photosynthesis is an essential pathway providing the chemical energy and reducing equivalents that sustain higher plant metabolism. It relies on sunlight, which is an inconstant source of energy that fluctuates in both intensity and spectrum. The fine and rapid tuning of the photosynthetic apparatus is essential to cope with changing light conditions and increase plant fitness. Recently PROTON GRADIENT REGULATION 6 (PGR6-ABC1K1), an atypical plastoglobule-associated kinase, was shown to regulate a new mechanism of light response by controlling the homeostasis of photoactive plastoquinone (PQ). PQ is a crucial electron carrier existing as a free neutral lipid in the photosynthetic thylakoid membrane. Perturbed homeostasis of PQ impairs photosynthesis and plant acclimation to high light. Here we show that a homologous kinase, ABC1K3, which like PGR6-ABC1K1 is associated with plastoglobules, also contributes to the homeostasis of the photoactive PQ pool. Contrary to PGR6-ABC1K1, ABC1K3 disfavors PQ availability for photosynthetic electron transport. In fact, in the abc1k1/abc1k3 double mutant the pgr6(abc1k1) the photosynthetic defect seen in the abc1k1 mutant is mitigated. However, the PQ concentration in the photoactive pool of the double mutant is comparable to that of abc1k1 mutant. An increase of the PQ mobility, inferred from the kinetics of its oxidation in dark, contributes to the mitigation of the pgr6(abc1k1) photosynthetic defect. Our results also demonstrate that ABC1K3 contributes to the regulation of other mechanisms involved in the adaptation of the photosynthetic apparatus to changes in light quality and intensity such as the induction of thermal dissipation and state transitions. Overall, we suggests that, besides the absolute concentration of PQ, its mobility and exchange between storage and active pools are critical for light acclimation in plants.

Essential role for phytol kinase and tocopherol in tolerance to combined light and temperature stress in tomato.

In a changing environment, plants need to cope with the impact of rising temperatures together with high light intensity. Here, we used lipidomics in the tomato model system to identify lipophilic molecules that enhance tolerance to combined high-temperature and high-light stress. Among several hundred metabolites, the two most strongly up-regulated compounds were α-tocopherol and plastoquinone/plastoquinol. Both are well-known lipid antioxidants and contribute to the protection of photosystem II (PSII) against photodamage under environmental stress. To address the protective function of tocopherol, an RNAi line (vte5) with decreased expression of VTE5 and reduced levels of α-tocopherol was selected. VTE5 encodes phytol kinase, which acts in the biosynthetic pathway of tocopherols. vte5 suffered strong photoinhibition and photobleaching when exposed to combined high-light and high-temperature stress, but neither stress alone produced a visible phenotype. As vte5 had plastoquinone levels similar to those of the wild type under combined stress, the strong phenotype could be attributed to the lack of α-tocopherol. These findings suggest that VTE5 protects against combined high-light and high-temperature stress and does so by supporting α-tocopherol production.