Probing the in situ volumes of Arabidopsis leaf plastids using three‐dimensional confocal and scanning electron microscopy

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm3. Wild-type guard cell plastids are approximately 18 μm3. Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment

Genetically Encoded Biosensors in Plants: Pathways to Discovery.

Genetically encoded biosensors that directly interact with a molecule of interest were first introduced more than 20 years ago with fusion proteins that served as fluorescent indicators for calcium ions. Since then, the technology has matured into a diverse array of biosensors that have been deployed to improve our spatiotemporal understanding of molecules whose dynamics have profound influence on plant physiology and development. In this review, we address several types of biosensors with a focus on genetically encoded calcium indicators, which are now the most diverse and advanced group of biosensors. We then consider the discoveries in plant biology made by using biosensors for calcium, pH, reactive oxygen species, redox conditions, primary metabolites, phytohormones, and nutrients. These discoveries were dependent on the engineering, characterization, and optimization required to develop a successful biosensor; they were also dependent on the methodological developments required to express, detect, and analyze the readout of such biosensors.Gatsby Research Fellowship awarded to A.M.J

Plant Immune Memory in Systemic Tissue Does Not Involve Changes in Rapid Calcium Signaling

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses

Genetically Encoded Biosensors in Plants: Pathways to Discovery.

Genetically encoded biosensors that directly interact with a molecule of interest were first introduced more than 20 years ago with fusion proteins that served as fluorescent indicators for calcium ions. Since then, the technology has matured into a diverse array of biosensors that have been deployed to improve our spatiotemporal understanding of molecules whose dynamics have profound influence on plant physiology and development. In this review, we address several types of biosensors with a focus on genetically encoded calcium indicators, which are now the most diverse and advanced group of biosensors. We then consider the discoveries in plant biology made by using biosensors for calcium, pH, reactive oxygen species, redox conditions, primary metabolites, phytohormones, and nutrients. These discoveries were dependent on the engineering, characterization, and optimization required to develop a successful biosensor; they were also dependent on the methodological developments required to express, detect, and analyze the readout of such biosensors.Gatsby Research Fellowship awarded to A.M.J

Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs.

The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by "monomeric" PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA

GFP-GEF1 is relocated to the prevacuolar compartment (PVC) in response to ABA treatment.

<p>(A) Co-localization assays of GFP-GEF1 with the indicated organelle markers in <i>N</i>. <i>benthamiana</i> leaf epidermal cells in response to ABA. GFP-GEF1 was co-expressed with mCherry-labeled organelle markers in <i>N</i>. <i>benthamiana</i> leaf epidermal cells. <i>N</i>. <i>benthamiana</i> leaves were treated with 50 μM ABA for 1 h before confocal imaging. Co-localization is visible as yellow dots in the merged images (Merge). Organelle marker names are listed in parentheses on the left. PVC, pre-vacuolar compartment. Representative images are shown of three independent co-localization experiments. (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002461#sec016" target="_blank">Methods</a>). Yellow boxes indicate approximate regions amplified to the right of merged images. Levels of co-localization for yellow boxed regions are depicted in relative intensity (<i>x-</i>, <i>y</i>-axes) scatter plots. Values of the linear Pearson correlation coefficient (rp) and the non-linear Spearman’s rank (rs) correlation coefficient were calculated and are given in the upper left corner of scatter plots. (B) Confocal images of the subcellular localization of GFP-GEF1 in response to the indicated treatments. 4-day-old <i>Arabidopsis</i> seedlings overexpressing GFP-GEF1 incubated with 50 μM ABA only (left), ABA plus 20 μM Wortmannin (PI3K inhibitor, disrupts protein trafficking into PVC) (middle), or ABA plus 0.1% (v/v) DMSO (Wortmannin solvent) (right) for 1 h before confocal imaging. Yellow boxes indicate regions of magnified images to the right of boxed images. Short yellow arrows point to ring-like structures typically induced by Wortmannin treatment. Scale bars 10 μm.</p

GEF1 protein is degraded in response to ABA treatment.

<p>Western blot analyses of GFP-GEF1 protein levels in response to the indicated treatments. Ten-day-old <i>pUBQ-GFP-GEF1 Arabidopsis</i> seedlings were immersed in 1/2 MS liquid medium for 1 h, then transferred into 1/2 MS medium supplemented with 50 μM ABA or 50 μM ABA plus 20 μM Wortmannin, and 0.1% (v/v) EtOH for the indicated durations. For MG132 treatment, seedlings were immersed in 1/2 MS medium with 50 μM MG132 for 2 h, then transferred in 1/2 MS medium with 50 μM ABA plus 50 μM MG132 for the indicated durations. Total protein extracts (20 μg) were subjected to immunoblot analysis with GFP antibody. Coomassie blue staining of SDS-polyacrylamide gel (SDS-PAGE) was used as a loading control.</p

GEF1 directly interacts with <i>Arabidopsis</i> type 2C protein phosphatase ABI1.

<p>(A) Yeast-two-hybrid (Y2H) assay of interactions of GEF1 with the indicated PP2Cs. The indicated construct combinations were co-transformed into the yeast strain pJ69-4A. Transformants were grown on-L-W control plates (left) for 3 d and -L-W-H (lacking Leucine, Tryptophan, and Histidine) selective plates with 3 mM 3-amino-1,2,4-triazole (3-AT) (right) for 6 d. The interactions of ABI1/PYL9 and ROP11/GEF1 were used as positive controls, and ABI1/PYL1 without ABA as negative control. (B) In vitro binding assay of GEF1 and ABI1. Each input protein was incubated with Glutathione Sepharose beads containing GST-GEF1 or GST control protein, followed by GST-GEF1 and GST affinity purification and subsequent immunoblotting with anti-Strep-II antibody. Bait (left): Coomassie brilliant blue staining of purified bait protein GST-GEF1 and GST; Pull-down: western blot of pull-down with Gluothathione Sepharose and probed with anti-Strep-II antibody; Input: Coomassie brilliant blue staining of recombinant input proteins StrepII-ABI1, StrepII-OST1, and StrepII-ROP11. ROP11/GEF1 and OST1/GEF1 binding were used as positive and negative controls, respectively.</p