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Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis

ABSTRACT: The bundle sheath cells (BSCs) layer – a presumed control point for radial transport of water and solutes between the vasculature and the leaf mesophyll cells (MCs) – is still largely understudied. Using isolated protoplasts, we found that 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, suggesting unique functionality of membrane transport in the BSCs, supported also by functional assays (electrophysiology and fluorescence imaging). A tight control of long distance transport is required for the optimization of the hydro-mineral homeostasis of organs in plants under fluctuating ambient conditions. The mechanism of ion selectivity and absorption from the xylem to the leaf is still poorly understood. The bundle sheath (BS), tightly enwrapping the leaf vasculature, has been suggested as a part of this mechanism, acting as a selective barrier which regulates the radial transport of water and solutes from the xylem to leaf cells. This suggestion relies on the anatomy, as well as on the recent physiological transport assays of the BS and its cells (BSCs). We hypothesized that the unique transport functionality of the BSCs is manifested in its transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) of Arabidopsis thaliana leaves. Indeed, in conformation of our hypothesis, 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, with the proton-pump AHA2 as a prominent example. Moreover, fluorescence imaging using the potentiometric dye (di-8 ANEPPS) revealed a more negative membrane potential of the BSCs protoplasts compared to those of MCs, and electrophysiological assays (patch-clamp) showed that the major, AKT2-like, membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Combined, these differences are compatible with an expected possibility of simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs in otherwise similar conditions.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE85463 | GEO | 2017/10/22

SECONDARY ACCESSION(S): PRJNA338594

REPOSITORIES: GEO

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Project description:A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. Keywords: Gene expression profiling of bundle sheath and mesophyll cell types

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Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis

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