Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration

Khoshravesh, Roxana; Lundsgaard-Nielsen, Vanessa; Sultmanis, Stefanie; Sage, Tammy L.

doi:10.1007/978-1-4939-7225-8_17

Cited by 17 publications

(13 citation statements)

References 20 publications

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“…Leaves 4 and 7 were prepared for light and transmission electron microscopy (TEM), quantification of cellular features, and immunodetection of photosynthetic enzymes and glycine decarboxylase as described previously [ 59 ]. 2 mm leaf sections from the middle of recent fully expanded leaves were fixed in 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 6.8) overnight at room temperature, and post-fixed (2 hr) in 1% osmium tetroxide.…”

Section: Methodsmentioning

confidence: 99%

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

et al. 2017

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SummaryThe C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called “proto-Kranz” anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in “proto-Kranz” species, and, as such, a key step toward engineering C4 rice has been achieved.

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Section: Methodsmentioning

confidence: 99%

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

et al. 2017

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“…The youngest cohort of fully expanded leaves was sampled in full sun for all procedures. Samples were fixed in 0.5% (v/v) glutaraldehyde, 4% (w/v) paraformaldehyde in sodium cacodylate buffer for light and transmission electron microscopy (TEM), and embedded in Spurr's resin and London Resin White as described by Khoshravesh et al (2017). Immunocytochemical detection of GLDP and Rubisco LSU followed Khoshravesh et al (2016Khoshravesh et al ( , 2017 with the same antibodies used for the immunoblots (Supplemental Fig.…”

Section: Leaf Anatomy Ultrastructure and Immunolocalizationmentioning

confidence: 99%

The Evolutionary Origin of C₄ Photosynthesis in the Grass Subtribe Neurachninae

et al. 2019

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The Australian grass subtribe Neurachninae contains closely related species that use C 3 , C 4 , and C 2 photosynthesis. To gain insight into the evolution of C 4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C 3 -C 4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO 2 ) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO 2 is concentrated in C 2 and C 4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C 3 Neurachninae species with C * values of 56-61 mmol mol 21 , whereas twothirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C 2 phenotype with C * of 44 mmol mol 21 . Increased investment of GLDP in MS tissue of the C 2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C 4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO 2 in MS tissues. 566

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“…Internal leaf anatomy was assessed on sections sampled from the middle of the second leaf pair (one leaf per plant: three plants per line). Plants were sampled between 09:00 a.m. and 11:00 a.m. and prepared for light and transmission (TEM) microscopy as described by (Khoshravesh et al ., ). The resin blocks that contained leaf material with third vein order were chosen for sectioning.…”

Section: Methodsmentioning

confidence: 97%

Reporter‐based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

et al. 2019

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SummaryThe evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre‐condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4–like bundle sheath. To this end, an ethylmethanesulfonate (EMS)‐based forward genetic screen was established in the Brassicaceae C3 species Arabidopsis thaliana. To ensure a high‐throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast‐targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping‐by‐sequencing approach the genomic segments that contained mutated candidate genes were identified.

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Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration

Cited by 17 publications

References 20 publications

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

The Evolutionary Origin of C₄ Photosynthesis in the Grass Subtribe Neurachninae

Reporter‐based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

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Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration

Cited by 17 publications

References 20 publications

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae

Reporter‐based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

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The Evolutionary Origin of C₄ Photosynthesis in the Grass Subtribe Neurachninae