An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells

Oscillations in cytosolic-free Ca²⁺ concentration ([Ca2+]_i) have been proposed to encode information that controls stomatal closure. [Ca2+]_i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca2+]_i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca2+]_i and, with the accompanying oscillations in voltage, drive the K⁺ and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.Stomata in the leaf epidermis are the main pathway both for CO₂ entry for photosynthesis and for foliar water loss by transpiration. Guard cells surround the stomatal pore and regulate the aperture, balancing the often conflicting demands for CO₂ and water conservation. Guard cells open and close the pore by expanding and contracting through the uptake and loss, respectively, of osmotic solutes, notably of K⁺, Cl⁻, and malate²⁻ (Mal²⁻; Pandey et al., 2007; Kim et al., 2010; Roelfsema and Hedrich, 2010; Lawson and Blatt, 2014). These transport processes comprise the final effectors of a regulatory network that coordinates transport across the plasma membrane and tonoplast, and maintains the homeostasis of the guard cell. A number of well-defined signals—including light, CO₂, drought and the water stress hormone abscisic acid (ABA)—act on this network, altering transport, solute content, turgor and cell volume, and ultimately stomatal aperture.Much research has focused on stomatal closure, underscoring both Ca²⁺-independent and Ca²⁺-dependent signaling. Of the latter, elevated cytosolic-free Ca²⁺ concentration ([Ca2+]_i) inactivates inward-rectifying K⁺ channels (I_K,in) to prevent K⁺ uptake and activates Cl⁻ (anion) channels (I_Cl) at the plasma membrane to depolarize the membrane and engage K⁺ efflux through outward-rectifying K⁺ channels (I_K,out; Keller et al., 1989; Blatt et al., 1990; Thiel et al., 1992; Lemtiri-Chlieh and MacRobbie, 1994). ABA, and most likely CO₂ (Kim et al., 2010), elevate [Ca2+]_i by facilitating Ca²⁺ entry at the plasma membrane to trigger Ca²⁺ release from endomembrane stores, a process often described as Ca²⁺-induced Ca²⁺ release (Grabov and Blatt, 1998, 1999). The hormone promotes Ca²⁺ influx by activating Ca²⁺ channels (I_Ca) at the plasma membrane, even in isolated membrane patches (Hamilton et al., 2000, 2001), which is linked to reactive oxygen species (Kwak et al., 2003; Wang et al., 2013). In parallel, cADP-ribose and nitric oxide promote endomembrane Ca²⁺ release and [Ca2+]_i elevation (Leckie et al., 1998; Neill et al., 2002; Garcia-Mata et al., 2003; Blatt et al., 2007). Best estimates indicate that endomembrane release accounts for more than 95% of the Ca²⁺ entering the cytosol to raise [Ca2+]_i (Chen et al., 2012; Wang et al., 2012).One feature of stomatal response to ABA, and indeed to a range of stimuli both hormonal as well as external, is its capacity for oscillations both in membrane voltage and [Ca2+]_i. Guard cell [Ca2+]_i at rest is typically around 100 to 200 nm, as it is in virtually all living cells. In response to ABA, [Ca2+]_i can rise above 1 μm—and locally, most likely above 10 μm—often in cyclic transients of tens of seconds to several minutes’ duration in association with oscillations in voltage and stomatal closure (Gradmann et al., 1993; McAinsh et al., 1995; Webb et al., 1996; Grabov and Blatt, 1998, 1999; Staxen et al., 1999; Allen et al., 2001). In principle, cycling in voltage and [Ca2+]_i arises as closure is accelerated with a controlled release of K⁺, Cl⁻, and Mal²⁻ from the guard cell and is subject to extracellular ion concentrations (Gradmann et al., 1993; Chen et al., 2012). However, it has been proposed that these, and similar oscillations in a variety of plant cell models, serve as physiological signals in their own right (McAinsh et al., 1995; Ehrhardt et al., 1996; Taylor et al., 1996). In support of such a signaling role, experiments designed to impose [Ca2+]_i (and voltage) oscillations in guard cells have yielded an optimal frequency for closure with a period near 10 min (Allen et al., 2001). Nonetheless, the studies offer no mechanistic explanation for this optimum that could validate a causal role in signaling, and none has been forthcoming since. Here we address questions of how such optimal frequencies in [Ca2+]_i oscillation arise and their relevance for stomatal closure, using quantitative systems analysis of guard cell transport and homeostasis. Our findings indicate that oscillations in voltage and [Ca2+]_i, and their optima associated with stomatal closure, are most simply explained as emerging from the interactions between ion transporters that drive stomatal closure. Thus, we conclude that these oscillations do not control, but are a by-product of the transport that determines stomatal aperture.     

Transforming ER exit: protein secretion meets oncogenesis

COPII-coated vesicles drive protein export from the endoplasmic reticulum (ER), although the regulation of this event, both spatially and kinetically, remains unclear. TFG is now defined as a factor that modulates recruitment of the coat and links ER sequestration of kinases to oncogenesis.     

Equal proportions of affected cells in muscle and blood of a mosaic carrier of facioscapulohumeral muscular dystrophy

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of D4Z4 repeat on 4q35. It displays a remarkable inter- and intra-familial clinical variability ranging from severe phenotype to asymptomatic carriers. Mosaicism for the contracted FSHD-sized allele is a recurrent finding, but only DNA from lymphocytes had been studied. It is currently not known if mosaicism is unequally distributed between different tissues and if muscle is relatively spared for the presence of the disease allele in mosaic asymptomatic carriers of a disease allele. Here we compare DNA extracted from peripheral blood lymphocytes (PBL), fibroblasts and muscle from a mosaic asymptomatic female carrier and mother of a FSHD patient. PFGE analysis showed a complex allelic segregation: two independent mitotic rearrangement episodes occurred, resulting in mosaicism for a contracted D4Z4 repeat on 4q35 in the mother and mosaicism for an expanded D4Z4 repeat on 10q26 in the affected daughter. The results show that the proportion of mosaicism in PBL and muscle were comparable, while in fibroblasts there was some variation in the mosaicism, which might be caused by culturing artefacts. This finding supports the hypothesis that a mitotic contraction of D4Z4 is an early embryonic event and indicates that the degree of mosaicism in PBL is representative for that of muscle.     

Prolyl endopeptidase and thrombin inhibitory diterpenoids from the bark of Xylopia aethiopica

The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and alpha-thrombin. Five compounds exhibited inhibitory activity against them.     

Automatic measurement of stomatal density from microphotographs

Key message

An automated process using a cascade classifier allowed the rapid assessment of the density and distribution of stomata on microphotographs from leaves of two oak species.

Abstract

Stomatal density is the number of stomata per unit area, an intensively studied trait, involved in the control of CO₂ and H₂O exchange between leaf and atmosphere. This trait is usually estimated by counting manually each stoma on a given surface (e.g., a microphotograph), usually repeating the procedure with images from different parts of the leaf. To improve this procedure, we tested the performance of a cascade classifier to automatically detect stomata on microphotographs from two oak species: Quercus afares Pomel and Quercus suber L. The two species are phylogenetically close with similar stomatal morphology, which allowed testing the reuse of the cascade classifier on stomata with similar shape. The results showed that a cascade classifier trained on only 100 sample views of stomata from Q. afares was able to rapidly detect stomata in Q. afares as well as in Q. suber with a very low number of false positives (5 %/1.9 %) and a small number of undetected stomata (14.8 %/0.74 %), when partial stomata near the edge of the microphotographs were ignored. The remaining undetected stomata were due to obstacles such as trichomes. As an example of further applications, we used the positions detected by the cascade classifier to assess the spatial distribution of stomata and group them on the leaf surface. To our knowledge this is the first time that a cascade classifier has been applied to plant microphotographs, and we were able to show that it can dramatically decrease the time needed to estimate stomatal density and spatial distribution.     

Functional Rescue of a Misfolded Eukaryotic ATP-binding Cassette Transporter by Domain Replacement

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.     

Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p,a Eukaryotic Drug Transporter

ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-ΔF670, similar to the predominant disease-causing allele in humans, CFTR-ΔF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-ΔF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and ΔF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.     

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.