The Relationships between Xylem Safety and Hydraulic Efficiency in the Cupressaceae: The Evolution of Pit Membrane Form and Function

Water transport in conifers occurs through single-celled tracheids that are connected to one another via intertracheid pit membranes. These membranes have two components: the porous margo, which allows water to pass through the membrane, and the impermeable torus, which functions to isolate gas-filled tracheids. During drought, tracheids can become air filled and thus hydraulically dysfunctional, a result of air entering through the pit membrane and nucleating cavitation in the water column. What are the hydraulic tradeoffs associated with cavitation resistance at the pit level, and how do they vary within the structural components of the intertracheid pit? To address these questions, we examined pit structure in 15 species of Cupressaceae exhibiting a broad range of cavitation resistances. Across species, cavitation resistance was most closely correlated to the ratio of the torus to pit aperture diameter but did not vary systematically with margo porosity. Furthermore, our data indicate that constraints on pit hydraulic efficiency are shared: the pit aperture limits pit conductivity in more drought-resistant taxa, while increased margo resistance is more likely to control pit conductivity in species that are more vulnerable to cavitation. These results are coupled with additional data concerning pit membrane structure and function and are discussed in the context of the evolutionary biogeography of the Cupressaceae.Water transport in conifers occurs through narrow, single-celled conduits (tracheids) that are organized in overlapping, longitudinal files. This simple, homoxylous arrangement represents an ancestral vascular design that has remained remarkably consistent since its first appearance in the progymnosperms of the Mid-Devonian (Taylor et al., 2009). However, the small size of tracheids can impose a high resistance to water transport as compared with the large, hydraulically efficient vessels present in many angiosperms (Hacke et al., 2004; Sperry et al., 2006). Despite this handicap, conifer tracheids can be just as hydraulically efficient as angiosperm xylem for a given conduit diameter, a result that can be wholly attributed to the distinctive structure of the conifer intertracheid pit membrane (Pittermann et al., 2005; Sperry et al., 2006).Because pit membranes also function to limit the spread of air from one conduit to another (cavitation), the physiological consequences of the transport efficiency versus cavitation safety tradeoffs in conifer and angiosperm pit membranes have received considerable attention at the pit and xylem levels, whereby cavitation resistance in north temperate woody plants appears to come at the cost of hydraulic efficiency (Pittermann et al., 2006a, 2006b; Sperry et al., 2006; Choat et al., 2008; Domec et al., 2008; Jansen et al., 2009; Schoonmaker et al., 2010). Previous work has shown that the integrated vascular performance of plants is key to understanding species distributions (Sperry et al., 1994; Brodribb and Hill, 1999; Pockman and Sperry, 2000; Choat et al., 2007), and within this framework, pit membranes have the potential to act as the nexus of the cavitation safety versus transport efficiency compromise. Yet, despite our progress, we are just starting to learn how these tradeoffs play out at the level of the pit membrane, particularly in one as complex as that of conifers. Hence, the goals of this study were to determine whether selection has acted to optimize conifer pit membrane performance in a manner that reflects species cavitation resistance and habitat distribution as well as to examine the role, if any, of evolutionary lineage.Unlike the homogenous pit membrane of angiosperm vessels, the conifer pit membrane is composed of two distinct regions: a thickened, centrally located torus and a porous margo region that surrounds it (Fig. 1; for study species, see Hacke et al., 2004; Choat et al., 2008; Choat and Pittermann, 2009). When tracheids are water filled, the pit membrane is centrally located in the pit chamber and water moves from tracheid to tracheid through the margo. Should an air-seeding event (cavitation) occur, causing a tracheid to become air filled, (i.e. embolized), the negative xylem pressure in the water-filled tracheid will act on the air-water interface in the margo pores by deflecting the pit membrane in the direction of the functional tracheid, thereby appressing the torus against the pit aperture border (Bailey, 1913; Liese, 1965; Liese and Bauch, 1967; Petty, 1972). This valve action of the membrane can create an effective seal that prevents further spread of air in the xylem. Cavitation is thought to occur when the water potential of the water-filled tracheid becomes negative enough to dislodge the torus from its sealing position, allowing air to enter the conduit. Overall, the structure of the torus-margo pit membrane must optimize what at first glance appear to be conflicting functional requirements: on the one hand, cavitation resistance selects for a combination of large tori and small apertures, but on the other hand, hydraulic efficiency favors porous margos, large apertures, and small tori.Open in a separate windowFigure 1.SEM images of intertracheid pit membranes belonging to nine Cupressaceae species (of 15) that represent the broad range of observed cavitation pressures. The opaque torus region of the membrane (T) is held centrally by the microfibrils of the margo (M). Visually, increased cavitation resistance appears to be associated with increased margo porosity, but quantitative estimates of margo resistance made on the most intact regions of the pit membranes (Fig. 8) revealed no differences among the species surveyed.

Table I.

Study species, figure abbreviations (Fig. Abbrevs.), locations (SFBG, San Francisco Botanical Garden, San Francisco; UCBG, University of California Botanical Garden, Berkeley, CA; UCSC, University of California, Santa Cruz, Arboretum, Santa Cruz, CA), and species natural history (Farjon, 2005)

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.     

Glc7-Reg1 phosphatase signals to Yck1,2 casein kinase 1 to regulate transport activity and glucose-induced inactivation of Saccharomyces maltose permease

The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1delta yck2-2(ts) (yck(ts)) strain with conditional Yck activity. Moreover, we provide evidence that Glc7-Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yck(ts) strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yck(ts) strain and maltose permease proteolysis is blocked. We show that a reg1delta mutant exhibits a phenotype remarkably similar to that conferred by yck(ts). The reg1delta phenotype is not enhanced in the yck(ts) reg1delta double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1delta mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1delta strain.     

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.     

Phenotypic anchoring of arsenic and cadmium toxicity in three hepatic-related cell systems reveals compound- and cell-specific selective up-regulation of stress protein expression: implications for fingerprint profiling of cytotoxicity

Exposure of cells to toxic chemicals is known to up-regulate the expression of a number of stress proteins (SPs), including metallothionein (MT) and members of the heat shock protein (HSP) family, and this response may allow the development of a fingerprint profile to identify mechanisms of toxicity in an in vitro toxicology setting. To test this hypothesis, three hepatic-derived cell culture systems (rat hepatoma FGC4 cell line, rat hepatocytes, human hepatoma HepG2 cell line) were exposed to cadmium (as CdCl2) and arsenic (as NaAsO2), two compounds believed to exert their toxicity through an oxidative stress mechanism, under conditions of phenotypic anchoring defined as minimal and mild toxicity (approximately 5 and 25% reduction in neutral red uptake, respectively). The expression of six SPs--MT, HSP25/27, HSP40, HSP60, HSP70, and HSP90--was then determined by ELISA. Expression of four of these SPs--MT, HSP25/27, HSP40 and HSP70--was up-regulated in at least one experimental condition. However, the patterns of expression of these four SPs varied across the experimental conditions, according to differences in toxicant concentration and/or level of toxicity, cell-type and toxicant itself. This lack of uniformity in response of a focussed set of mechanistically defensible targets suggests that similar problems may emerge when using more global approaches based on genomics and proteomics, in which problems of redundancy in targets and uncertain mechanistic relevance will be greater.     

PRENATAL GENETIC TESTING KITS SOLD AT YOUR LOCAL PHARMACY: PROMOTING AUTONOMY OR PROMOTING CONFUSION?

Research groups around the world are developing non‐invasive methods of prenatal genetic diagnosis, in which foetal cells are obtained by maternal blood test. Meanwhile, an increasing number of genetic tests are sold directly to the public. I extrapolate from these developments to consider a scenario in which PNGD self‐testing kits are sold directly to the public. Given the opposition to over‐the‐counter genetic tests and the continuing controversy surrounding PNGD, it is reasonable to expect objections to PNGD self‐testing kits. I focus on one potential objection, that PNGD self‐testing kits would undermine the autonomy of potential test subjects. More specifically, that ‘direct to the public’ PNGD would fail to ensure that consumers exercise autonomy in the following PNGD‐related choices:

• ? Should I use PNGD?
• ? Based on the results of the PNGD test, should I continue or terminate my pregnancy?

Under the current system, PNGD is provided by health care practitioners, who are required to counsel women both before and after the test. In contrast, ‘direct to the public’ PNGD would allow women to make their PNGD‐related decisions outside the context of the health care system. I compare these two decision‐making contexts, arguing that the health care system is not unequivocally better at promoting the autonomy of potential test subjects. Therefore the promotion of autonomy does not constitute a strong argument against such test kits. Other objections may be more persuasive, so I do not offer an overall assessment of the acceptability of ‘direct to the public’ PNGD.     

Molecular basis of inherited skin-blistering disorders, and therapeutic implications

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.     

The competent sentinel node: an association with an axillary presentation and an occult or a small primary invasive breast carcinoma

The concept of the sentinel node describes a primary or sentinel lymph node (SLN), which exists and through which tumour cells from a primary tumour in a particular location must first travel to spread to a particular regional lymph node group. In this series we present three patients presenting with a pathological axillary node associated with either an occult or very small primary breast cancer. In each case the primary tumour was found to have metastasised to the palpable node, however despite the significant enlargement of this node, no other axillary nodes were found to be affected on axillary node clearance. This has led us to postulate that the SLN in some cases contains unique characteristics that enable it to prevent further spread of the tumour up the lymphatic chain. Hence the term the competent sentinel node.