Preparation and applications of <i>Arabidopsis thaliana</i> guard cell protoplasts

There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance. KeywordsAbscissic acid sensitive, drought tolerance, stomatal behavior, vacuolar invertase inhibitor History

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“…Isolation of Arabidopsis guard cells was isolated from rosette leaves following the protocol described by Pandey et al 9 .…”

Section: Vacuolar Invertase Activity Analysis Of Guard Cellsmentioning

confidence: 99%

Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis

Chen

Liang

Yin

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…Guard cell protoplasts from tomato plants were isolated and purified mainly as described in the protocol developed for Arabidopsis thaliana (Pandey et al, 2002;Yoo et al, 2007) with modifications. Fully expanded leaves (;10 g) with the main veins removed were surfaced sterilized in 0.5% (v/v) NaOCl and 0.12% (v/v) Tween 20 solution for 5 min, washed in 96% (v/v) ethanol for 2 s, followed by three washes in sterile distilled water.…”

Section: Isolation Of Guard Cell Protoplastsmentioning

confidence: 99%

Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture

et al. 2011

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Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell-specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.

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“…However, GC are much smaller and comprise a minor fraction of the total cells in a leaf. Although guard cell protoplasts have been isolated mostly at a small scale from several plant species (12)(13)(14)(15)(16), it is often technically challenging to obtain GC with good quantity and good quality. With a full genome sequenced, Arabidopsis has become a model species for plant biology research.…”

mentioning

confidence: 99%

“…With a full genome sequenced, Arabidopsis has become a model species for plant biology research. The large scale guard cell preparation method (5,15) has great potential to enhance functional genomics of guard cell functions. The Assmann laboratory at the Pennsylvania State University started guard cell proteomics work several years ago and have identified more than 1800 unique guard cell proteins in Arabidopsis.…”

mentioning

confidence: 99%

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Zhu

Dai

McClung

et al. 2009

Molecular & Cellular Proteomics

111

115

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Guard cells (GC)1 are highly specialized cells that form tiny pores called stomata on the leaf surface. When environmental conditions change, guard cells can rapidly change shape so that the pores open or close to control leaf gas exchange and water transpiration. Mesophyll cells (MC) are mainly parenchyma cells between the upper and lower epidermis specialized for photosynthesis. Previous studies that focused on guard cell metabolism and response to environmental signals have revealed important features of functional differentiation of GC (1, 2). Compared with MC, GC contain few chloroplasts with very limited structures and thus possess very low photosynthetic capability. The Calvin cycle in GC only assimilates 2-4% of CO 2 fixed in MC (3). In contrast, GC contain abundant mitochondria and display a high respiratory rate, suggesting that oxidative phosphorylation is an important source of ATP to fuel the guard cell machinery (4). Using microarrays covering just one-third of the Arabidopsis genome, Leonhardt et al. (5) observed a differential abscisic acid (ABA) modulation of many guard cell ABA signaling components as well as key enzymes involved in carbon metabolism in GC and MC. This only available large scale genomics study identified 1309 guard cell expressed genes of which 64 transcripts mainly involved in transcription, signaling, and cytoskeleton were preferentially expressed in GC compared with MC. However, functional grouping of the genes revealed only a 1.9% higher representation of photosynthesis genes in MC than in GC. The percentages of genes in all other categories such as protein turnover, defense, signaling, channels and transporters, and metabolism are similar between the two distinct cell types (5). These proteins are known to play specific roles in guard cell functions (6). This highlights the necessity of studying guard cell functions at the protein level.To date, there have been very few analyses of single celltype proteomes in plants. Proteome analyses of trichomes from Arabidopsis (7) and tobacco (8) and root hairs from soybean (9) exist but have identified fewer than 100 proteins per proteome. The proteomes of pollen from different species have been relatively well studied, but the pollen grains are not single cells because they contain two/three-cell gametophytes (10). A critical factor for large scale proteomics analFrom the ‡Department

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Preparation and applications of Arabidopsis thaliana guard cell protoplasts

Cited by 80 publications

References 60 publications

Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis

Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis

Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

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