Membrane topology and sequence requirements for oil body targeting of oleosin

Oleosin protein is targeted to oil bodies via the endoplasmic reticulum (ER) and consists of a lipid-submerged hydrophobic (H) domain that is flanked by cytosolic hydrophilic domains. We investigated the relationship between oleosin ER topology and its subsequent ability to target to oil bodies. Oleosin variants were created to yield differing ER membrane topologies and tagged with a reporter enzyme. Localisation was assessed by fractionation after transient expression in embryonic cells. Membrane-straddled topologies with N-terminal sequence in the ER lumen and C-terminal sequence in the cytosol were unable to target to oil bodies efficiently. Similarly, a translocated topology with only ER membrane and lumenal sequence was unable to target to oil bodies efficiently. Both topology variants accumulated proportionately higher in ER microsomal fractions, demonstrating a block in transferring from ER to oil bodies. The residual oil body accumulation for the inverted topology was shown to be because of partial adoption of native ER membrane topology, using a reporter variant, which becomes inactivated by ER-mediated glycosylation. In addition, the importance of H domain sequence for oil body targeting was assessed using variants that maintain native ER topology. The central proline knot motif (PKM) has previously been shown to be critical for oil body targeting, but here the arms of the H domain flanking this motif were shown to be interchangeable with only a moderate reduction in oil body targeting. We conclude that oil body targeting of oleosin depends on a specific ER membrane topology but does not require a specific sequence in the H domain flanking arms.     

Ionic control of the lateral exchange of water between vascular bundles in tomato

Ions can enhance water flow through the xylem via changes in the hydraulic resistance at border pit membranes. Because flow between adjacent xylem vessels occurs primarily via bordered pit fields, it is hypothesized that xylem sap ion concentrations would affect lateral movement of water more than longitudinal flow. Using tomato as a model system, evidence is presented for ion-mediated changes in xylem hydraulic resistance and the lateral transport of water. Water flow between adjacent xylem bundles increased by approximately 50% in the presence of ions while longitudinal flow only increased by approximately 20%. However, the enhancement of lateral exchange due to ions was magnified by the presence of a pressure difference between vascular bundles. These results indicate that the degree of nutrient-sharing among sectors of a plant may depend on both nutrient concentration and the availability of water in the root zone.     

The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods

A comparison was made of three methods for measuring the leaf lamina hydraulic conductance (K(lamina)) for detached mature leaves of six woody temperate angiosperm species. The high-pressure method, the evaporative flux method and the vacuum pump method involve, respectively, pushing, evaporating and pulling water out of the lamina while determining the flow rate into the petiole and the water potential drop across the leaf. Tests were made of whether the high-pressure method and vacuum pump method measurements of K(lamina) on single leaves were affected by irradiance. In Quercus rubra, the high pressure method was sensitive to irradiance; K(lamina) measured under high irradiance (>1200 micro mol m(-2) s(-1 )photosynthetically active radiation) was 4.6-8.8 times larger than under ambient laboratory lighting (approximately 6 micro mol m(-2) s(-1 )photosynthetically active radiation). By constrast, the vacuum pump method was theoretically expected to be insensitive to irradiance, and this expectation was confirmed in experiments on Hedera helix. When used in the ways recommended here, the three methods produced measurements that agreed typically within 10%. There were significant differences in species' K(lamina); values ranged from 1.24x10(-4) kg s(-1) m(-2) MPa(-1) for Acer saccharum to 2.89x10(-4) kg s(-1) m(-2) MPa(-1) for Vitis labrusca. Accurate, rapid determination of K(lamina) will allow testing of the links between K(lamina), water-use, drought tolerance, and the enormous diversity of leaf form, structure and composition.     

Tolerization as a tool for generating novel monoclonal antibodies

Standard hybridoma production involves the fusion of spleen cells from an immunized mouse with a non-secretory murine myeloma cell line. While this technology has provided numerous reagents that are highly valuable, demand is now increasing for monoclonal antibodies which can distinguish between closely related antigens. Induction of tolerance towards common antigens enables the recovery of high-specificity reagents that have previously proved elusive. This review details a number of strategies using either complex protein mixtures or purified proteins as tolerogens and subsequent immunization with a closely related immunogen.     

Understanding the Hydraulics of Porous Pipes: Tradeoffs Between Water Uptake and Root Length Utilization

The water uptake region in roots is several hundred times longer than the root diameter. The distributed nature of the uptake zone requires that the hydraulic design of roots be understood by analogy to flow through a porous pipe. Here we present results of an analytical and experimental investigation that allowed an in-depth analysis of root hydraulic properties. Measurements on nodal maize roots confirm the nonlinear distribution of water uptake predicted by the porous pipe model. The major design parameter governing the distribution of water uptake along a porous pipe is the ratio between its axial and radial hydraulic resistance. However, total flow is proportional to the pipe's overall resistance. These results suggest the existence of a tradeoff between the effective utilization of root length and the total capacity for water uptake.     

Pigment dynamics and autumn leaf senescence in a New England deciduous forest,eastern USA

The leaves of woody plants at Harvard Forest in Central Massachusetts, USA, changed color during senescence; 70% (62/89) of the woody species examined anatomically contained anthocyanins during senescence. Anthocyanins were not present in summer green leaves, and appeared primarily in the vacuoles of palisade parenchyma cells. Yellow coloration was a result of the unmasking of xanthophyll pigments in senescing chloroplasts. In nine red-senescing species, anthocyanins were not detectable in mature leaves, and were synthesized de novo in senescence, with less than 20µg cm^–2 of chlorophyll remaining. Xanthophyll concentrations declined in relation to chlorophyll to the same extent in both yellow- and red-leaved taxa. Declines in the maximum photosystemII quantum yield of leaves collected prior to dawn were only slightly less in the red-senescing species, indicating no long-term protective activity. Red-leaved species had significantly greater mass/area and lower chlorophylla/b ratios during senescence. Nitrogen tissue concentrations in mature and senescent leaves negatively correlated to anthocyanin concentrations in senescent leaves, weak evidence for more efficient nitrogen resorption in anthocyanic species. Shading retarded both chlorophyll loss and anthocyanin production in Cornus alternifolia, Acer rubrum, Acer saccharum, Quercus rubra and Viburnum alnifolium. It promoted chlorophyll loss in yellow-senescing Fagus grandifolia. A reduced red:far-red ratio did not affect this process. Anthocyanins did not increase leaf temperatures in Q.rubra and Vaccinium corymbosum on cold and sunny days. The timing of leaf-fall was remarkably constant from year to year, and the order of senescence of individual species was consistent.     

Loss of HuR is linked to reduced expression of proliferative genes during replicative senescence

Cellular aging is accompanied by alterations in gene expression patterns. Here, using two models of replicative senescence, we describe the influence of the RNA-binding protein HuR in regulating the expression of several genes whose expression decreases during senescence. We demonstrate that HuR levels, HuR binding to target mRNAs encoding proliferative genes, and the half-lives of such mRNAs are lower in senescent cells. Importantly, overexpression of HuR in senescent cells restored a "younger" phenotype, while a reduction in HuR expression accentuated the senescent phenotype. Our studies highlight a critical role for HuR during the process of replicative senescence.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

Bordered pit structure and vessel wall surface properties. Implications for embolism repair

The idea that embolized xylem vessels can be refilled while adjacent vessels remain under tension is difficult to accept if the cavitated vessels remain hydraulically connected to vessels under tension. A mechanism by which embolized conduits could be hydraulically isolated from adjacent conduits requires the existence of a non-zero contact angle and a flared opening into the bordered pit chamber such that a convex air-water interface forms at the entrance into the pit chamber. We measured the contact angle and pit chamber geometry for six species. The contact angle measured in the vessel lumen ranged between 42 degrees to 55 degrees, whereas the opening into the pit chamber ranged between 144 degrees and 157 degrees. If the surface properties within the pit chamber are similar to those in the lumen, a convex meniscus will form at the flared opening into the pit chamber. The maximum pressure difference between water in the lumen and gas in the pit chamber that could be stabilized by this interface was calculated to be within the range of 0.07 to 0.30 MPa.     

Stem water storage and diurnal patterns of water use in tropical forest canopy trees

Stem water storage capacity and diurnal patterns of water use were studied in five canopy trees of a seasonal tropical forest in Panama. Sap flow was measured simultaneously at the top and at the base of each tree using constant energy input thermal probes inserted in the sapwood. The daily stem storage capacity was calculated by comparing the diurnal patterns of basal and crown sap flow. The amount of water withdrawn from storage and subsequently replaced daily ranged from 4 kg d^–1 in a 0·20-m-diameter individual of Cecropia longipes to 54 kg d^–1 in a 1·02-m-diameter individual of Anacardium excelsum, representing 9–15% of the total daily water loss, respectively. Ficus insipida, Luehea seemannii and Spondias mombin had intermediate diurnal water storage capacities. Trees with greater storage capacity maintained maximum rates of transpiration for a substantially longer fraction of the day than trees with smaller water storage capacity. All five trees conformed to a common linear relationship between diurnal storage capacity and basal sapwood area, suggesting that this relationship was species-independent and size-specific for trees at the study site. According to this relationship there was an increment of 10 kg of diurnal water storage capacity for every 0·1 m² increase in basal sapwood area. The diurnal withdrawal of water from, and refill of, internal stores was a dynamic process, tightly coupled to fluctuations in environmental conditions. The variations in basal and crown sap flow were more synchronized after 1100 h when internal reserves were mostly depleted. Stem water storage may partially compensate for increases in axial hydraulic resistance with tree size and thus play an important role in regulating the water status of leaves exposed to the large diurnal variations in evaporative demand that occur in the upper canopy of seasonal lowland tropical forests.