PECTIN METHYLESTERASE34 Contributes to Heat Tolerance through Its Role in Promoting Stomatal Movement.

Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then is partially demethylesterified by pectin methylesterases (PMEs; EC 3.1.1.11). PME activity on the status of pectin methylesterification profoundly affects the properties of pectin and, thereby, is critical for plant development and the plant defense response, although the roles of PMEs under heat stress (HS) are poorly understood. Functional genome annotation predicts that at least 66 potential PME genes are contained in Arabidopsis (Arabidopsis thaliana). Thermotolerance assays of PME gene T-DNA insertion lines revealed two null mutant alleles of PME34 (At3g49220) that both consistently showed reduced thermotolerance. Nevertheless, their impairment was independently associated with the expression of HS-responsive genes. It was also observed that PME34 transcription was induced by abscisic acid and highly expressed in guard cells. We showed that the PME34 mutation has a defect in the control of stomatal movement and greatly altered PME and polygalacturonase (EC 3.2.1.15) activity, resulting in a heat-sensitive phenotype. PME34 has a role in the regulation of transpiration through the control of the stomatal aperture due to its cell wall-modifying enzyme activity during the HS response. Hence, PME34 is required for regulating guard cell wall flexibility to mediate the heat response in Arabidopsis.

The Heat Stress Factor HSFA6b Connects ABA Signaling and ABA-Mediated Heat Responses.

Heat stress response (HSR) is a conserved mechanism developed to increase the expression of heat shock proteins (HSPs) via a heat shock factor (HSF)-dependent mechanism. Signaling by the stress phytohormone abscisic acid (ABA) is involved in acquired thermotolerance as well. Analysis of Arabidopsis (Arabidopsis thaliana) microarray databases revealed that the expression of HSFA6b, a class A HSF, extensively increased with salinity, osmotic, and cold stresses, but not heat. Here, we show that HSFA6b plays a pivotal role in the response to ABA and in thermotolerance. Salt-inducible HSFA6b expression was down-regulated in ABA-insensitive and -deficient mutants; however, exogenous ABA application restored expression in ABA-deficient, but not -insensitive plants. Thus, ABA signaling is required for proper HSFA6b expression. A transcriptional activation assay of protoplasts revealed that ABA treatment and coexpression of an ABA signaling master effector, ABA-RESPONSIVE ELEMENT-BINDING PROTEIN1, could activate the HSFA6b promoter. In addition, HSFA6b directly bound to the promoter of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2A and enhanced its expression. Analysis of ABA responses in seed germination, cotyledon greening, and root growth as well as salt and drought tolerance in HSFA6b-null, overexpression, and dominant negative mutants revealed that HSFA6b is a positive regulator participating in ABA-mediated salt and drought resistance. Thermoprotection tests showed that HSFA6b was required for thermotolerance acquisition. Our study reveals a network in which HSFA6b operates as a downstream regulator of the ABA-mediated stress response and is required for heat stress resistance. This new ABA-signaling pathway is integrated into the complex HSR network in planta.

How ambient temperature affects the heading date of foxtail millet (Setaria italica).

Foxtail millet (Setaria italica), a short-day plant, is one of the important crops for food security encountering climate change, particularly in regions where it is a staple food. Under the short-day condition in Taiwan, the heading dates (HDs) of foxtail millet accessions varied by genotypes and ambient temperature (AT). The allelic polymorphisms in flowering time (FT)-related genes were associated with HD variations. AT, in the range of 13°C-30°C that was based on field studies at three different latitudes in Taiwan and observations in the phytotron at four different AT regimes, was positively correlated with growth rate, and high AT promoted HD. To elucidate the molecular mechanism of foxtail millet HD, the expression of 14 key FT-related genes in four accessions at different ATs was assessed. We found that the expression levels of SiPRR95, SiPRR1, SiPRR59, SiGhd7-2, SiPHYB, and SiGhd7 were negatively correlated with AT, whereas the expression levels of SiEhd1, SiFT11, and SiCO4 were positively correlated with AT. Furthermore, the expression levels of SiGhd7-2, SiEhd1, SiFT, and SiFT11 were significantly associated with HD. A coexpression regulatory network was identified that shown genes involved in the circadian clock, light and temperature signaling, and regulation of flowering, but not those involved in photoperiod pathway, interacted and were influenced by AT. The results reveal how gene × temperature and gene × gene interactions affect the HD in foxtail millet and could serve as a foundation for breeding foxtail millet cultivars for shift production to increase yield in response to global warming.

Effect of prolonged mechanical ventilation on cough function and TRPV1 expression.

Cough is a pivotal airway protective reflex, yet the effects of prolonged mechanical ventilation (PMV) on cough function are unknown. This study compared the cough function in subjects with PMV (≥ 21 days, n = 29) and those with short-term mechanical ventilation (SMV, ≤ 7 days, n = 27). Cough reflex sensitivity was measured by capsaicin provocation concentrations after extubation. The cough strength of respiratory muscles was assessed by involuntary cough peak expiratory flow (iCPEF). The mRNA expression of transient receptor potential vanilloid 1 (TRPV1), a cough sensor activated by capsaicin, in tracheal tissues was determined. We found that cough reflex sensitivity and iCPEF were significantly lower in the PMV group than in the SMV group. The tracheal expression of TRPV1 was similar in both groups, suggesting that changes in TRPV1 expression may not be a contributing factor. Our finding regarding the cough dysfunction after PMV highlights the need to implement effective airway clearance management and rehabilitation in this population.

Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis.

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.

Immunoexpression and prognostic role of hTERT and cyclin D1 in urothelial carcinoma.

The aim was to investigate the expression of human telomerase reverse transcriptase (hTERT) and cyclin D1 in correlation with clinicopathologic features of urothelial carcinoma (UC). Tissue microarrays (TMA) were constructed from paraffin-embedded specimens of 94 UC patients and immunohistochemical staining was used. High hTERT expression was found in 50 (53%) of the 94 tumors and was significantly associated with tumor invasiveness and tumor grade (P=0.008 and 0.0190, respectively). High cyclin D1 expression was found in 69 (73%) of the 94 tumors and was significantly associated with non-invasiveness and smaller tumor size, but there was no correlation with tumor grade. Kaplan-Meier analysis indicated that patients with low hTERT and high cyclin D1 levels had longer local recurrence-free survival (P=0.0482 and 0.0123, respectively). In addition, patients with high cyclin D1 levels had longer disease-free survival (P=0.0195). In conclusion, this study demonstrated that hTERT and cyclin D1 may be of recurrence predictive value for UC, thus providing clinicians with ancillary information when deciding on suitable therapeutic strategies in UC.

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia.

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,ß-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17ß-estradiol (E2) at a low concentration (30 µg/ml) but not at high concentrations (50 and 150 µg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density.

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation. As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope.

Pectin methylesterase is required for guard cell function in response to heat.

Pectin is an important cell wall polysaccharide required for cellular adhesion, extension, and plant growth. The pectic methylesterification status of guard cell walls influences the movement of stomata in response to different stimuli. Pectin methylesterase (PME) has a profound effect on cell wall modification, especially on the degree of pectic methylesterification during heat response. The Arabidopsis thaliana PME34 gene is highly expressed in guard cells and in response to the phytohormone abscisic acid. The genetic data highlighted the significant role of PME34 in heat tolerance through the regulation of stomatal movement. Thus, the opening and closure of stomata is mediated by changes in response to a given stimulus, could require a specific cell wall modifying enzyme to function properly.

Three novel alleles of FLOURY ENDOSPERM2 (FLO2) confer dull grains with low amylose content in rice.

Rice is a major food source for much of the world, and expanding our knowledge of genes conferring specific rice grain attributes will benefit both farmer and consumer. Here we present novel dull grain mutants with a low amylose content (AC) derived from mutagenesis of Oryza sativa, ssp. japonica cv. Taikeng 8 (TK8). Positional cloning of the gene conferring the dull grain phenotype revealed a point mutation located at the acceptor splice site of intron 11 of FLOURY ENDOSPERM2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein. Three novel flo2 alleles were identified herein, which surprisingly conferred dull rather than floury grains. The allelic diversity of flo2 perturbed the expression of starch synthesis-related genes including OsAGPL2, OsAGPS2b, OsGBSSI, OsBEI, OsBEIIb, OsISA1, and OsPUL. The effect of the flo2 mutations on the physicochemical properties of the grain included a low breakdown, setback, and consistency of rice, indicating a good elasticity and soft texture of cooked rice grains. The effects of FLO2, combined with the genetic background of the germplasm and environmental effects, resulted in a variety of different amylose content levels, grain appearance, and physicochemical properties of rice, providing a host of useful information to future grain-quality research and breeding.

Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of OsADF3 confers Arabidopsis Thaliana drought tolerance.

BACKGROUND: Actin depolymerizing factors (ADFs) are small actin-binding proteins. Many higher-plant ADFs has been known to involve in plant growth, development and pathogen defense. However, in rice the temporal and spatial expression of OsADF gene family and their relationship with abiotic stresses tolerance is still unknown. RESULTS: Here we reported the first comprehensive gene expression profile analysis of OsADF gene family. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We also demonstrated that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3 were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was enhanced in lateral roots and root tips. Ectopically overexpressed OsADF3 conferred the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, primary root length and survival. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were upregulated in transgenic Arabidopsis under drought stress. CONCLUSIONS: These results suggested that OsADF gene family may participate in plant abiotic stresses response or tolerance and would facilitate functional validation of other OsADF genes.

Autocrine and paracrine regulation of interleukin-8 expression in lung cancer cells.

We had previously demonstrated that lung cancer cells, upon contact with macrophages, could be induced to secrete angiogenic factors to promote tumor angiogenesis. In this study, we focused on the paracrine and autocrine regulation of interleukin (IL)-8 expression in sensitized lung cancer cells after interacting with macrophages. We found that the IL-8 mRNA expression in lung cancer cells significantly increased after coculture with phorbol myristate acetate-treated THP-1 cells and human primary lung macrophages. Fresh lung cancer CL1-5 cells cocultured with macrophage-sensitized lung cancer cells still had a 35% of increase in IL-8 mRNA expression. The addition of anti-inflammatory agents pyrrolidine dithiocarbamate, pentoxifylline, aspirin, and dexamethasone could completely suppress the expression of IL-8 mRNA in fresh/sensitized lung cancer cell cocultures. Human recombinant tumor necrosis factor (TNF)-alpha and IL-1alpha could induce IL-8 expression in lung cancer cells in a dose-dependent manner. Neutralization with TNF-alpha and IL-1alpha antibodies in cocultures decreased the levels of IL-8 expression in sensitized lung cancer cells. Nuclear factor-kappaB transcriptional activity was also suppressed by the same antibodies, as confirmed by a reporter gene assay and the electrophoretic mobility shift assay. Our results highly suggest that both autocrine and paracrine regulation are involved in IL-8 expression of lung cancer cells cocultured with macrophage. Also, the regulations of IL-8 expression in lung cancer cells were through the nuclear factor-kappaB pathway and modulated by TNF-alpha and IL-1alpha.