Fluorescent Protein-Based Approaches for Subcellular Protein Localization in Plants

Sharma, Mayank; Klösgen, Ralf Bernd; Bennewitz, Bationa

doi:10.1007/978-1-0716-2667-2_9

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…Subcellular localization of the researched proteins helps us to understand their functions, and currently transient expression of recombinant fluorescent proteins in plants is a common method to detect the localization of target proteins in plant cells because transient expression techniques are not affected by gene silencing and have more stable expression efficiency and higher translation rates [ 37 – 39 ]. In this study, subcellular localization analysis revealed that AaCaM3 localized on the cytoplasm and nucleus, the result is similar to what predicted by the online prediction software TargetP1.1 and is also largely similar to the localization of CaM3 in other plants.…”

Section: Discussionmentioning

confidence: 99%

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus

Niu,

Zhou,

Yue

et al. 2024

BMC Plant Biol

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Amorphophallus is a perennial monocotyledonous herbaceous plant native to the southwestern region of China, widely used in various fields such as food processing, biomedicine and chemical agriculture. However, Amorphophallus is a typical thermolabile plant, and the continuous high temperature in summer have seriously affected the growth, development and economic yield of Amorphophallus in recent years. Calmodulin (CaM), a Ca2+ sensor ubiquitous in eukaryotes, is the most important multifunctional receptor protein in plant cells, which affects plant stress resistance by participating in the activities of a variety of signaling molecules. In this study, the key gene AaCaM3 for the Ca2+-CaM regulatory pathway was obtained from A. albus, the sequence analysis confirmed that it is a typical calmodulin. The qRT-PCR results demonstrated that with the passage of heat treatment time, the expression of AaCaM3 was significantly upregulated in A. albus leaves. Subcellular localization analysis revealed that AaCaM3 localized on the cytoplasm and nucleus. Meanwhile, heterologous transformation experiments have shown that AaCaM3 can significantly improve the heat tolerance of Arabidopsis under heat stress. The promoter region of AaCaM3 was sequenced 1,338 bp by FPNI-PCR and GUS staining assay showed that the promoter of AaCaM3 was a high-temperature inducible promoter. Yeast one-hybrid analysis and Luciferase activity reporting system analysis showed that the AaCaM3 promoter may interact with AaHSFA1, AaHSFA2c, AaHSP70, AaDREB2a and AaDREB2b. In conclusion, this study provides new ideas for further improving the signal transduction network of high-temperature stress in Amorphophallus.

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Section: Discussionmentioning

confidence: 99%

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus

Niu,

Zhou,

Yue

et al. 2024

BMC Plant Biol

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“…Binary plant expression vectors were transferred into the Agrobacterium tumefaciens strain GV3101 (pMP90) ( 25 ). Transient transformation of Nicotiana benthamiana leaf tissue was performed as described previously ( 26 ). Arabidopsis plants were transformed using a floral dip method as described by Davis et al 27 .…”

Section: Methodsmentioning

confidence: 99%

MFP1 defines the subchloroplast location of starch granule initiation

Sharma,

Abt,

Eicke

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants “mis”-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, “re” localizing MFP1 to the stromal face of the chloroplast’s inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

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“…Agrobacterium-mediated transient transformation of N. benthamiana leaf tissue was performed as described previously (59). The transiently transformed leaf cells were analyzed using a confocal system, LSM780 from Zeiss using either laser of 514 nm (YFP), 458 nm (CFP) and 633 nm (chlorophyll) wavelengths.…”

Section: Confocal Laser Scanning Microscopy (Clsm)mentioning

confidence: 99%

A conserved ESCRT-II-like protein participates in the biogenesis and maintenance of thylakoid membranes

Yilmazer,

Vetrano,

Eicke

et al. 2023

Preprint

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Thylakoids are membrane-bound compartments located in cyanobacteria and chloroplasts of plants and algae. They play an indispensable role in the light-driven reactions that enable photosynthetic organisms to convert water and carbon dioxide into oxygen and sugars. The biogenesis and maintenance of thylakoid membranes is a critical yet underappreciated area of research. One of the few known critical regulators of this process, VIPP1 (Vesicle-Inducing Protein in Plastids 1), was recently shown to be structurally similar to ESCRT-III proteins-the first evidence for ESCRT-like (Endosomal Sorting Complex Required for Transport) machinery in chloroplasts. Here, we used an affinity purification approach in two distantly related photosynthetic eukaryotes, the green alga Chlamydomonas reinhardtii and the plant Arabidopsis thaliana, to discover proteins that interact with VIPP1. Among several newly identified proteins, we focused on a highly conserved but uncharacterized protein (VIPP1-Associated protein 1, VIA1) that robustly interacts with VIPP1 in both systems. VIA1 is predicted to contain a winged-helix domain, a characteristic feature of ESCRT-II proteins that mediates the interaction with ESCRT-III proteins. The absence of VIA1 causes thylakoid swelling upon exposure to high light in Chlamydomonas and defective thylakoid biogenesis in the newly emerging leaf tissue in Arabidopsis, thereby delaying chloroplast development in this tissue. We propose that VIA1 is part of a previously unrecognized chloroplast ESCRT-like system that plays a critical role in forming, remodeling, and repairing photosynthetic membranes.

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Fluorescent Protein-Based Approaches for Subcellular Protein Localization in Plants

Cited by 3 publications

References 18 publications

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus

MFP1 defines the subchloroplast location of starch granule initiation

A conserved ESCRT-II-like protein participates in the biogenesis and maintenance of thylakoid membranes

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