Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification,while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1 driven by the promoter from CELLULOSE SYNTHASE7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.     

Vaccine-Induced Boosting of Influenza Virus-Specific CD4 T Cells in Younger and Aged Humans

Current yearly influenza virus vaccines induce strain-specific neutralizing antibody (NAb) responses providing protective immunity to closely matched viruses. However, these vaccines are often poorly effective in high-risk groups such as the elderly and challenges exist in predicting yearly or emerging pandemic influenza virus strains to include in the vaccines. Thus, there has been considerable emphasis on understanding broadly protective immunological mechanisms for influenza virus. Recent studies have implicated memory CD4 T cells in heterotypic immunity in animal models and in human challenge studies. Here we examined how influenza virus vaccination boosted CD4 T cell responses in younger versus aged humans. Our results demonstrate that while the magnitude of the vaccine-induced CD4 T cell response and number of subjects responding on day 7 did not differ between younger and aged subjects, fewer aged subjects had peak responses on day 14. While CD4 T cell responses were inefficiently boosted against NA, both HA and especially nucleocaspid protein- and matrix-(NP+M) specific responses were robustly boosted. Pre-existing CD4 T cell responses were associated with more robust responses to influenza virus NP+M, but not H1 or H3. Finally pre-existing strain-specific NAb decreased the boosting of CD4 T cell responses. Thus, accumulation of pre-existing influenza virus-specific immunity in the form of NAb and cross-reactive T cells to conserved virus proteins (e.g. NP and M) over a lifetime of exposure to infection and vaccination may influence vaccine-induced CD4 T cell responses in the aged.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.     

Performance of a new dynamic model for predicting diurnal time courses of stomatal conductance at the leaf level

Under natural conditions, plants are subjected to continuous changes of irradiance that drive variations of stomatal conductance to water vapour (g_s). We propose a dynamic model to predict the temporal response of g_s at the leaf level using an asymmetric sigmoid function with a unique parameter describing time constants for increasing and decreasing g_s. The model parameters were adjusted to observed data using Approximate Bayesian Computation. We tested the model performance for (1) instant changes of irradiance; or (2) continuous and controlled variations of irradiance simulating diurnal time courses. Compared with the two mostly used steady‐state models, our dynamic model described daily time courses of g_s with a higher accuracy. In particular, it was able to describe the hysteresis of g_s responses to increasing/decreasing irradiance and the resulting rapid variations of intrinsic water‐use efficiency. Compared to the mechanistic model of temporal responses of g_s by Kirschbaum, Gross & Pearcy, for which time constants were estimated with a large variance, our model estimated time constants with a higher precision. It is expected to improve predictions of water loss and water‐use efficiency in higher scale models by using a small number of parameters.     

Elevated Temperature and CO2 Stimulate Late-Season Photosynthesis But Impair Cold Hardening in Pine

Rising global temperature and CO₂ levels may sustain late-season net photosynthesis of evergreen conifers but could also impair the development of cold hardiness. Our study investigated how elevated temperature, and the combination of elevated temperature with elevated CO₂, affected photosynthetic rates, leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening in Eastern white pine (Pinus strobus). We designed an experiment where control seedlings were acclimated to long photoperiod (day/night 14/10 h), warm temperature (22°C/15°C), and either ambient (400 μL L⁻¹) or elevated (800 μmol mol⁻¹) CO₂, and then shifted seedlings to growth conditions with short photoperiod (8/16 h) and low temperature/ambient CO₂ (LTAC), elevated temperature/ambient CO₂ (ETAC), or elevated temperature/elevated CO₂ (ETEC). Exposure to LTAC induced down-regulation of photosynthesis, development of sustained nonphotochemical quenching, accumulation of soluble carbohydrates, expression of a 16-kD dehydrin absent under long photoperiod, and increased freezing tolerance. In ETAC seedlings, photosynthesis was not down-regulated, while accumulation of soluble carbohydrates, dehydrin expression, and freezing tolerance were impaired. ETEC seedlings revealed increased photosynthesis and improved water use efficiency but impaired dehydrin expression and freezing tolerance similar to ETAC seedlings. Sixteen-kilodalton dehydrin expression strongly correlated with increases in freezing tolerance, suggesting its involvement in the development of cold hardiness in P. strobus. Our findings suggest that exposure to elevated temperature and CO₂ during autumn can delay down-regulation of photosynthesis and stimulate late-season net photosynthesis in P. strobus seedlings. However, this comes at the cost of impaired freezing tolerance. Elevated temperature and CO₂ also impaired freezing tolerance. However, unless the frequency and timing of extreme low-temperature events changes, this is unlikely to increase risk of freezing damage in P. strobus seedlings.Land surface temperature is increasing, particularly in the northern hemisphere (IPCC, 2014), which is dominated by boreal and temperate forests. At higher latitudes, trees rely on temperature and photoperiod cues to detect changing seasons and to trigger cessation of growth and cold hardening during the autumn (Ensminger et al., 2015). For boreal and temperate evergreen conifers, cold hardening involves changes in carbohydrate metabolism, down-regulation of photosynthesis, accumulation of cryoprotective metabolites, and development of freezing tolerance (Crosatti et al., 2013; Ensminger et al., 2015). These processes minimize freezing damage and enable conifers to endure winter stresses. However, rising temperatures result in asynchronous phasing of temperature and photoperiod characterized by delayed arrival of first frosts (McMahon et al., 2010), which may impact the onset and development of cold hardening during autumn.Short photoperiod induces the cessation of growth in many tree species (Downs and Borthwick, 1956; Heide, 1974; Repo et al., 2000; Böhlenius et al., 2006). As a consequence, carbon demand in sink tissue decreases toward the end of the growing season, and the bulk of photoassimilate is translocated from source tissues to storage tissues (Hansen and Beck, 1994; Oleksyn et al., 2000). In addition, cryoprotective soluble sugars, including sucrose, raffinose, and pinitol, accumulate in leaf tissues to enhance freezing tolerance (Strimbeck et al., 2008; Angelcheva et al., 2014). Thus, by winter, leaf nonstructural carbohydrates are mainly comprised of mono- and oligosaccharides, and only minimal levels of starch remain (Hansen and Beck, 1994; Strimbeck et al., 2008). The concurrent decrease of photoassimilate and demand for metabolites that occur during the cessation of growth also impacts the citric acid cycle that mediates between photosynthesis, respiration, and protein synthesis. The citric acid cycle generates NADH to fuel ATP synthesis via mitochondrial electron transport, as well as amino acid precursors (Shi et al., 2015). In C3 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC) converts phosphoenolpyruvate to oxaloacetic acid in order to supplement the flow of metabolites to the citric acid cycle and thus controls the regulation of respiration and photosynthate partitioning (O’Leary et al., 2011).Cessation of growth, low temperature, and presumably short photoperiod decrease the metabolic sink for photoassimilates, resulting in harmful excess light energy (Öquist and Huner, 2003; Ensminger et al., 2006) and increased generation of reactive oxygen species (Adams et al., 2004). During autumn and the development of cold hardiness, conifers reconfigure the photosynthetic apparatus in order to avoid formation of excess light and reactive oxygen species. This involves a decrease in chlorophylls and PSII reaction center core protein D1 (Ottander et al., 1995; Ensminger et al., 2004; Verhoeven et al., 2009), as well as aggregation of light-harvesting complex proteins (Ottander et al., 1995; Busch et al., 2007). Additionally, photoprotective carotenoid pigments accumulate in leaves, especially the xanthophylls, zeaxanthin, and lutein that contribute to nonphotochemical quenching (NPQ) via thermal dissipation of excess light energy (Busch et al., 2007; Verhoeven et al., 2009; Demmig-Adams et al., 2012). Prolonged exposure to low temperature induces sustained nonphotochemical quenching (NPQ_S), where zeaxanthin constitutively dissipates excess light energy (Ensminger et al., 2004; Demmig-Adams et al., 2012; Fréchette et al., 2015).In conifers, freezing tolerance is initiated during early autumn in response to decreasing photoperiod (Rostad et al., 2006; Chang et al., 2015) and continues to develop through late autumn in response to the combination of short photoperiod and low temperature (Strimbeck and Schaberg, 2009; Chang et al., 2015). In addition to changes in carbohydrate content, freezing tolerance also involves the expression of specific dehydrins (Close, 1997; Kjellsen et al., 2013). Members of the dehydrin protein family are involved in responses to osmotic, salt, and freezing stress (Close, 1996). Dehydrins have been associated with improved freezing tolerance in many species including spinach (Kaye et al., 1998), strawberry (Houde et al., 2004), cucumber (Yin et al., 2006), peach (Wisniewski et al., 1999), birch (Puhakainen et al., 2004), and spruce (Kjellsen et al., 2013). In angiosperms, a characteristic Lys-rich dehydrin motif known as the K-segment interacts with lipids to facilitate membrane binding (Koag et al., 2003; Eriksson et al., 2011). Several in vitro studies have demonstrated dehydrin functions including prevention of aggregation and unfolding of enzymes (using Vitis riparia; Hughes and Graether, 2011), radical scavenging (using Citrus unshiu; Hara et al., 2004), and suppression of ice crystal formation (using Prunus persica; Wisniewski et al., 1999). To date, dehydrin functions have not been demonstrated in planta.Rising temperatures since the mid-twentieth century have delayed the onset of autumn dormancy and increased length of the growing season in forests across the northern hemisphere (Boisvenue and Running, 2006; Piao et al., 2007; McMahon et al., 2010). Studies have shown that elevated temperatures ranging from +4°C to +20°C above ambient can delay down-regulation of photosynthesis in several evergreen conifers. Consistent findings were apparent among climate-controlled chamber studies exposing Pinus strobus seedlings to a sudden shift in temperature and/or photoperiod (Fréchette et al., 2016), as well as chamber studies exposing Picea abies seedlings to simulated autumn conditions using a gradient of decreasing temperature and photoperiod (Stinziano et al., 2015). Similar findings were also demonstrated in open-top chamber experiments exposing mature Pinus sylvestris to a gradient of decreasing temperature and natural photoperiod (Wang, 1996). Elevated temperature (+4°C above ambient) also impaired cold hardening in Pseudotsuga menziesii seedlings (Guak et al., 1998) and mature P. sylvestris (Repo et al., 1996) exposed to a decreasing gradient of temperature and natural photoperiod using open-top chambers. In contrast, a recent study showed that smaller temperature increments (+1.5°C to +3°C) applied using infrared heaters did not delay down-regulation of photosynthesis or impair freezing tolerance in field-grown P. strobus seedlings that were acclimated to larger diurnal and seasonal temperature variations (Chang et al., 2015). For many tree species, photoperiod determines cessation of growth (Tanino et al., 2010; Petterle et al., 2013), length of the growing season (Bauerle et al., 2012), and development of cold hardiness (Welling et al., 1997; Li et al., 2003; Rostad et al., 2006). However, the effects of climate warming on tree phenology are complex and can be unpredictable due to species- and provenance-specific differences in sensitivity to photoperiod and temperature cues (Körner and Basler, 2010; Basler and Körner, 2012; Basler and Körner, 2014).The effect of elevated CO₂ further increases uncertainties in the response of trees to warmer climate. Similar to warmer temperature, elevated CO₂ may also delay the down-regulation of photosynthesis in evergreens and extend the length of the growing season, as demonstrated in mature P. sylvestris (Wang, 1996). Elevated CO₂ increases carbon assimilation (Curtis and Wang, 1998; Ainsworth and Long, 2005) and biomass production (Ainsworth and Long, 2005) during the growing season. The effects could continue during the autumn if dormancy or growth cessation is delayed, which suggests that elevated CO₂ may increase annual carbon uptake. However, long-term exposure to elevated CO₂ can also down-regulate photosynthesis during the growing season (Ainsworth and Long, 2005). Prior studies that have attempted to determine the impact of a combination of elevated CO₂ and/or temperature on cold hardening in evergreens have largely focused on freezing tolerance, with contrasting results. Open-top chamber experiments showed that a combination of elevated temperature and CO₂ both delayed and impaired freezing tolerance of P. menziesii seedlings (Guak et al., 1998) and evergreen broadleaf Eucalyptus pauciflora seedlings (Loveys et al., 2006) but did not affect freezing tolerance of mature P. sylvestris (Repo et al., 1996). A recent field experiment examining mature trees revealed that Larix decidua, but not Pinus mugo, exhibited enhanced freezing damage following six years of exposure to combined soil warming and elevated CO₂ (Rixen et al., 2012). In contrast, a climate-controlled study showed that exposure to elevated CO₂ advanced the date of bud set and improved freezing tolerance in Picea mariana seedlings (Bigras and Bertrand, 2006). In a second study on similar seedlings conducted by the same authors, exposure of trees to elevated CO₂ also enhanced freezing tolerance but impaired the accumulation of sucrose and raffinose (Bertrand and Bigras, 2006). These previous experiments used experimental conditions where temperature and photoperiod gradually decreased. While this approach aims to mimic natural conditions, it is difficult to distinguish specific responses to either photoperiod or temperature. Because of the contrasting findings from previous studies, we designed an experiment aiming to separate the effects of photoperiod, temperature, and CO₂ on a wide range of parameters that are involved in cold hardening in conifers.Our study aimed to determine (1) how induction and development of the cold hardening process is affected by a shift from long to short photoperiod under warm conditions and (2) how the combination of warm air temperature and elevated CO₂ affects photoperiod-induced cold hardening processes in Eastern white pine (P. strobus). To assess the development of cold hardening, we measured photosynthetic rates, changes in leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening over 36 d. Assuming that both low temperature and short photoperiod cues are required to induce cold hardening in conifers, we hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would prevent seedlings growing under autumn photoperiod from down-regulating photosynthesis. We further hypothesized that warm temperature and the combination of warm temperature and elevated CO₂ would impair the development of freezing tolerance, due to a lack of adequate phasing of the low temperature and short photoperiod signals.     

Neutrophil thrombospondin receptors are linked to GTP-binding proteins

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP-binding proteins (G-proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP-mediated motility. Both PT and CT inhibited TSP-mediated chemotaxis and substrate-associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 μM) nor 8-bromo-cAMP (300 μM) significantly affected TSP-mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 ± 0.3 to 14.2 ± 0.1 pmol/10⁶ cells). Although we could not test the PT sensitivity of TSP priming for FMLP-mediated chemotaxis (as PT inhibits FMLP-mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP-dependent priming of FMLP-mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G-proteins was obtained by examining the effect of TSP on α-subunit ADP-ribosylation, GTPase activity, and GTPγS binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT-dependent ribosylation of G_iα2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G-proteins. However, TSP did not block the CT-dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of G_iα2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 ± 2.7% of control values). In addition, GTPγS binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 ± 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G-proteins, a subpopulation of which also associates with FMLP receptors. © 1996 Wiley-Liss, Inc.     

IFN-gamma-dependent nitric oxide production is not linked to resistance in experimental African trypanosomiasis

Resistance to African trypanosomes is dependent on B cell and Th1 cell responses to the variant surface glycoprotein (VSG). While B cell responses to VSG control levels of parasitemia, the cytokine responses of Th1 cells to VSG appear to be linked to the control of parasites in extravascular tissues. We have recently shown that IFN-gamma knockout (IFN-gamma KO) mice are highly susceptible to infection and have reduced levels of macrophage activation compared to the wild-type C57BL/6 (WT) parent strain, even though parasitemias were controlled by VSG-specific antibody responses in both strains. In the present work, we examine the role of IFN-gamma in the induction of nitric oxide (NO) production and host resistance and in the development of suppressor macrophage activity in mice infected with Trypanosoma brucei rhodesiense. In contrast to WT mice, susceptible IFN-gamma KO mice did not produce NO during infection and did not develop suppressor macrophage activity, suggesting that NO might be linked to resistance but that suppressor cell activity was not associated with resistance or susceptibility to trypanosome infection. To further examine the consequence of inducible NO production in infection, we monitored survival, parasitemia, and Th cell cytokine production in iNOS KO mice. While survival times and parasitemia of iNOS KO mice did not differ significantly from WT mice, VSG-specific Th1 cells from iNOS KO mice produced higher levels of IFN-gamma and IL-2 than cells from WT mice. Together, these results show for the first time that inducible NO production is not the central defect associated with susceptibility of IFN-gamma KO mice to African trypanosomes, that IFNgamma-induced factors other than iNOS may be important for resistance to the trypanosomes, and that suppressor macrophage activity is not linked to either the resistance or the susceptibility phenotypes.     

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.