Measuring the Osmotic Water Permeability Coefficient (P&lt;sub&gt;f&lt;/sub&gt;) of Spherical Cells: Isolated Plant Protoplasts as an Example

Shatil‐Cohen, Arava; Sibony, Hadas; Chaumont, François; Moran, Nava; Moshelion, Menachem

doi:10.3791/51652

Cited by 17 publications

(32 citation statements)

References 17 publications

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“…This protoplast isolation procedure resulted in a very high yield of protoplasts (20 million protoplasts per gram of leaf tissue). For detailed description (video article) of the protoplast isolation and Pf measurement, please see Shatil-Cohen et al (2014).…”

Section: Protoplast Isolationmentioning

confidence: 99%

The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics

et al. 2014

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Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (K leaf ), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate K leaf . To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (P f ), mesophyll conductance of CO 2 , photosynthesis, K leaf , transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS P f and decreased K leaf but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced P f , but not reduced mesophyll conductance of CO 2 , suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed.

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Section: Protoplast Isolationmentioning

confidence: 99%

The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics

et al. 2014

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“…For a detailed description of the protoplast isolation and P f measurement, please see Shatil‐Cohen et al . ().…”

Section: Methodsmentioning

confidence: 97%

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential

et al. 2017

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Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration.

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“…Protoplasts were isolated from 6- to 8-week-old plants using the rapid method 60 . Briefly, the lower leaf epidermis was peeled off from the middle part of the leaf and the peeled leaves were cut into small squares and incubated in an enzyme solution containing 3.3% (w/w) of an enzyme mix comprised of the following enzymes in the given proportions: 0.55 g of cellulase (Worthington), 0.1 g of pectolyase (Karlan), 0.33 g of polyvinyl pyrrolidone K30 (Sigma-Aldrich) and 0.33 g of bovine serum albumin (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Vascular bundle sheath and mesophyll regulation of leaf water balance in response to chitin

Attia

Dalal

Moshelion

2018

Preprint

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Plants can detect pathogen invasion by sensing pathogen-associated molecular patterns (PAMPs). This sensing process leads to the induction of defense responses. Most PAMP mechanisms of action have been described in the guard cells. Here, we describe the effects of chitin, a PAMP found in fungal cell walls, on the cellular osmotic water permeability (P f ) of the leaf vascular bundle-sheath (BS) and mesophyll cells and its subsequent effect on leaf hydraulic conductance (K leaf ).The BS is a parenchymatic tissue that tightly encases the vascular system. BS cells have been shown to control K leaf through changes in their P f , for example, in response to ABA.It was recently reported that, in Arabidopsis, the chitin receptors chitin elicitor receptor kinase 1 (CERK1) and LYSINE MOTIF RECEPTOR KINASE 5 (LYK5) are highly expressed in the BS, as well as the neighboring mesophyll. Therefore, we studied the possible impact of chitin on these cells.Our results revealed that both BS cells and mesophyll cells exhibit a sharp decrease in P f in response to chitin treatment. In addition, xylem-fed chitin decreased K leaf and led to stomatal closure. However, an Atlyk5 mutant showed none of these responses.Complimenting AtLYK5 specifically in the BS cells (using the SCARECROW promoter) and transient expresion in mesophyll cells each resulted in a response to chitin that was similar to that observed in the wild type. These results suggest that BS and mesophyll cells each play a role in the perception of apoplastic chitin and in initiating chitintriggered immunity.Significance Statement: PAMP perception by plant receptors triggers various defense responses important for plant immunity. Here we provide new insights into a topic that has received a great deal of previous attention, revealing that a chitin immune response is present in additional leaf tissues other than the stomata. Chitin perception by the bundle sheath cells enwrapping the whole leaf vascular system decrease its cellular osmotic permeability and leaf hydraulic conductance. This in turn, leads to hydraulic signals 1 1

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Measuring the Osmotic Water Permeability Coefficient (P<sub>f</sub>) of Spherical Cells: Isolated Plant Protoplasts as an Example

Cited by 17 publications

References 17 publications

The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics

The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential

Vascular bundle sheath and mesophyll regulation of leaf water balance in response to chitin

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