Stomatal Responses to Flooding of the Intercellular Air Spaces Suggest a Vapor-Phase Signal Between the Mesophyll and the Guard Cells

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO₂. These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K⁺ in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.Stomatal responses to the environment have been studied in leaves for well over 100 years. More recently, the mechanisms for these responses have been investigated using isolated epidermes or isolated guard cell protoplasts. Despite the combination of these two approaches, the mechanisms by which stomata respond to environmental signals are not well understood. Since stomata control CO₂ uptake and water loss from leaves, the responses of stomata to environmental factors are important determinants of terrestrial productivity and water use. It is therefore critical that we understand the mechanisms by which stomata respond to the environment if we are to accurately predict the effects of future climates on productivity and water cycles (Randall et al., 1996).There are two assumptions about stomata that are implicit in much of the recent literature: (1) that stomatal responses result from sensory mechanisms that reside within the guard cells, and (2) that stomata in isolated epidermes respond similarly to those in a leaf. The exception to this generalization is the stomatal response to humidity, which has been suggested to be the result of changes in guard cell water potential (Dewar, 1995, 2002) or of signaling from other cells in the leaf to the guard cells (Buckley et al., 2003). The assumption that guard cells directly sense CO₂ and light is largely based on data from isolated epidermes that show effects of light and CO₂ on stomatal apertures. As pointed out by Mott (2009), however, stomatal responses to light and CO₂ in isolated epidermes are generally much different from those observed in leaves; e.g. responses in isolated epidermes are generally smaller than those in leaves, opening in response to light is slower, and closing in darkness is rarely observed. These observations were used to suggest that the mesophyll is somehow involved in stomatal responses to red light and CO₂. This idea is supported by several recent studies that suggest that guard cells do not respond directly to red light. In the first of these studies it was shown that guard cells in an intact leaf do not show hyperpolarization of the plasma membrane in response to red light if the red light is applied to only the guard cell (Roelfsema et al., 2002). In contrast, blue light applied only to the guard cell does cause hyperpolarization, and red light does cause hyperpolarization if applied to the guard cell and the underlying mesophyll. The second study showed that stomata in albino areas of a leaf do not respond to red light, although they contain chloroplasts and do respond to blue light (Roelfsema et al., 2006). Finally, a third study has shown that isolated epidermes are much more sensitive to light and CO₂ when placed in close contact with an exposed mesophyll from a leaf from the same or a different species (Mott et al., 2008). These epidermis-mesophyll grafts showed stomatal responses to light and CO₂ that were indistinguishable from those in an intact leaf—a sharp contrast to the behavior of stomata in isolated epidermes that are floating on buffer solutions. In that study, illumination of a single stoma in a leaf using a small-diameter fiber optic did not produce stomatal opening, but opening did occur if several stomata and the underlying mesophyll were illuminated. Furthermore, this treatment actually caused opening of adjacent, but unilluminated, stomata (Mott et al., 2008).In constructing the epidermis-mesophyll grafts in the study described above (Mott et al., 2008), it was noticed that functional grafts could be produced only if both the mesophyll and the epidermis were blotted completely dry of any free water before placing them together. Although the tissues were apparently still fully hydrated, there was very little free water present (i.e. water not contained within the walls of the leaf cells), and both the mesophyll and epidermis felt and looked dry prior to assembly. In addition, even when free water was blotted away initially, stomata did not open in grafts that ended up with visible water on the epidermis or mesophyll that was caused by condensation during the experiment. These observations suggest that the presence of free water somehow prevented the stomata in the grafts from opening. Assuming that the mechanisms operating in the grafts were similar to those in an intact leaf, this result also suggests that free water may have an effect on stomata in leaves as well. In addition, it seems possible that the effect of free water on stomata could be related to the disruption of the signal from the mesophyll that was proposed in an earlier study (Mott et al., 2008). We hypothesize that disruption of this signal could be caused by (1) dilution of some solute that is necessary for opening (such as K⁺) in the guard cell walls, (2) dilution of an apoplastic, liquid-phase opening signal from the mesophyll to the guard cells, and (3) blockage of a vapor-phase opening signal from the mesophyll to the guard cells. This study was initiated to test these three hypotheses by examining the effect of free water and other liquids on stomatal functioning.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation)

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1'' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

A molecular genetic linkage map identifying the St and H subgenomes of Elymus (Poaceae: Triticeae) wheatgrass

Elymus L. is the largest and most complex genus in the Triticeae tribe of grasses with approximately 150 polyploid perennial species occurring worldwide. We report here the first genetic linkage map for Elymus. Backcross mapping populations were created by crossing caespitose Elymus wawawaiensis (EW) (Snake River wheatgrass) and rhizomatous Elymus lanceolatus (EL) (thickspike wheatgrass) to produce F(1) interspecific hybrids that were then backcrossed to the same EL male to generate progeny with segregating phenotypes. EW and EL are both allotetraploid species (n = 14) containing the St (Pseudoroegneria) and H (Hordeum) genomes. A total of 387 backcross progeny from four populations were genotyped using 399 AFLP and 116 EST-based SSR and STS markers. The resulting consensus map was 2574 cM in length apportioned among the expected number of 14 linkage groups. EST-based SSR and STS markers with homology to rice genome sequences were used to identify Elymus linkage groups homoeologous to chromosomes 1-7 of wheat. The frequency of St-derived genome markers on each linkage group was used to assign genome designations to all linkage groups, resulting in the identification of the seven St and seven H linkage groups of Elymus. This map also confirms the alloploidy and disomic chromosome pairing and segregation of Elymus and will be useful in identifying QTLs controlling perennial grass traits in this genus.     

Using progenitor strain information to identify quantitative trait nucleotides in outbred mice

We have developed a fast and economical strategy for dissecting the genetic architecture of quantitative trait loci at a molecular level. The method uses two pieces of information: mapping data from crosses that involve more than two inbred strains and sequence variants in the progenitor strains within the interval containing a quantitative trait locus (QTL). By testing whether the strain distribution pattern in the progenitor strains is consistent with the observed genetic effect of the QTL we can assign a probability that any sequence variant is a quantitative trait nucleotide (QTN). It is not necessary to genotype the animals except at a skeleton of markers; the genotypes at all other polymorphisms are estimated by a multipoint analysis. We apply the method to a 4.8-Mb region on mouse chromosome 1 that contains a QTL influencing anxiety segregating in a heterogeneous stock and show that, under the assumption that a single QTN is present and lies in a region conserved between the human and mouse genomes, it is possible to reduce the number of variants likely to be the quantitative trait nucleotide from many thousands to <20.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.