Transcriptomic analysis of hub genes regulating albinism in light- and temperature-sensitive albino tea cultivars 'Zhonghuang 1' and 'Zhonghuang 2'.

Albino tea cultivars have high economic value because their young leaves contain enhanced free amino acids that improve the quality and properties of tea. Zhonghuang 1 (ZH1) and Zhonghuang 2 (ZH2) are two such cultivars widely planted in China; however, the environmental factors and molecular mechanisms regulating their yellow-leaf phenotype remain unclear. In this study, we demonstrated that both ZH1 and ZH2 are light- and temperature-sensitive. Under natural sunlight and low-temperature conditions, their young shoots were yellow with decreased chlorophyll and an abnormal chloroplast ultrastructure. Conversely, young shoots were green with increased chlorophyll and a normal chloroplast ultrastructure under shading and high-temperature conditions. RNA-seq analysis was performed for high light and low light conditions, and pairwise comparisons identified genes exhibiting different light responses between albino and green-leaf cultivars, including transcription factors, cytochrome P450 genes, and heat shock proteins. Weighted gene coexpression network analyses of RNA-seq data identified the modules related to chlorophyll differences between cultivars. Genes involved in chloroplast biogenesis and development, light signaling, and JA biosynthesis and signaling were typically downregulated in albino cultivars, accompanied by a decrease in JA-ILE content in ZH2 during the albino period. Furthermore, we identified the hub genes that may regulate the yellow-leaf phenotype of ZH1 and ZH2, including CsGDC1, CsALB4, CsGUN4, and a TPR gene (TEA010575.1), which were related to chloroplast biogenesis. This study provides new insights into the molecular mechanisms underlying leaf color formation in albino tea cultivars.

Two interacting ethylene response factors regulate heat stress response.

The ethylene response factor (ERF) transcription factors are integral components of environmental stress signaling cascades, regulating a wide variety of downstream genes related to stress responses and plant development. However, the mechanisms by which ERF genes regulate the heat stress response are not well understood. Here, we uncover the positive role of ethylene signaling, ERF95 and ERF97 in basal thermotolerance of Arabidopsis thaliana. We demonstrate that ethylene signaling-defective mutants exhibit compromised basal thermotolerance, whereas plants with constitutively activated ethylene response show enhanced basal thermotolerance. EIN3 physically binds to the promoters of ERF95 and ERF97. Ectopic constitutive expression of ERF95 or ERF97 increases the basal thermotolerance of plants. In contrast, erf95 erf96 erf97 erf98 quadruple mutants exhibit decreased basal thermotolerance. ERF95 and ERF97 genetically function downstream of EIN3. ERF95 can physically interact with ERF97, and this interaction is heat inducible. ERF95 and ERF97 regulate a common set of target genes, including known heat-responsive genes and directly bind to the promoter of HSFA2. Thus, our study reveals that the EIN3-ERF95/ERF97-HSFA2 transcriptional cascade may play an important role in the heat stress response, thereby establishing a connection between ethylene and its downstream regulation in basal thermotolerance of plants.

Recent advances and biotechnological applications of RNA metabolism in plant chloroplasts and mitochondria.

The chloroplast and mitochondrion are semi-autonomous organelles that play essential roles in cell function. These two organelles are embellished with prokaryotic remnants and contain many new features emerging from the co-evolution of organelles and the nucleus. A typical plant chloroplast or mitochondrion genome encodes less than 100 genes, and the regulation of these genes' expression is remarkably complex. The regulation of chloroplast and mitochondrion gene expression can be achieved at multiple levels during development and in response to environmental cues, in which, RNA metabolism, including: RNA transcription, processing, translation, and degradation, plays an important role. RNA metabolism in plant chloroplasts and mitochondria combines bacterial-like traits with novel features evolved in the host cell and is regulated by a large number of nucleus-encoded proteins. Among these, pentatricopeptide repeat (PPR) proteins are deeply involved in multiple aspects of the RNA metabolism of organellar genes. Research over the past decades has revealed new insights into different RNA metabolic events in plant organelles, such as the composition of chloroplast and mitochondrion RNA editosomes. We summarize and discuss the most recent knowledge and biotechnological implications of various RNA metabolism processes in plant chloroplasts and mitochondria, with a focus on the nucleus-encoded factors supporting them, to gain a deeper understanding of the function and evolution of these two organelles in plant cells. Furthermore, a better understanding of the role of nucleus-encoded factors in chloroplast and mitochondrion RNA metabolism will motivate future studies on manipulating the plant gene expression machinery with engineered nucleus-encoded factors.

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid's protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling.

During development or under stress, chloroplasts generate signals that regulate the expression of a large number of nuclear genes, a process called retrograde signaling. GENOMES UNCOUPLED 1 (GUN1) is an important regulator of this pathway. In this study, we have discovered an unexpected role for GUN1 in plastid RNA editing, as gun1 mutations affect RNA-editing efficiency at multiple sites in plastids during retrograde signaling. GUN1 plays a direct role in RNA editing by physically interacting with MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2). MORF2 overexpression causes widespread RNA-editing changes and a strong genomes uncoupled (gun) molecular phenotype similar to gun1 MORF2 further interacts with RNA-editing site-specificity factors: ORGANELLE TRANSCRIPT PROCESSING 81 (OTP81), ORGANELLE TRANSCRIPT PROCESSING 84 (OTP84), and YELLOW SEEDLINGS 1 (YS1). We further show that otp81, otp84, and ys1 single mutants each exhibit a very weak gun phenotype, but combining the three mutations enhances the phenotype. Our study uncovers a role for GUN1 in the regulation of RNA-editing efficiency in damaged chloroplasts and suggests that MORF2 is involved in retrograde signaling.

Analysis of ocular inflammation in anterior chamber-involving uveitis using swept-source anterior segment OCT.

PURPOSE: To evaluate the utility of swept-source (SS) optical coherence tomography (OCT) to objectively analyze the degree of anterior chamber (AC) inflammation. METHODS: Thirty-eight eyes of 32 patients with uveitis and 20 control eyes were enrolled. SS OCT B-scans were obtained, and the number of cells in the B-scans was counted using two methods: (1) manual grading by Point Picker plug-in of Image J ( http://bigwww.epfl.ch/thevenaz/pointpicker/ ) and (2) automated grading by the Image J Particle Analysis algorithm ( http://imagej.net/Particle_Analysis ). The automated and manual AC cell counts were correlated with the Standardization of Uveitis Nomenclature score. RESULTS: The average numbers of AC inflammatory cells counted by the automated method were 8 ± 4.0, 18 ± 3.0, 42 ± 14.0, 81 ± 32.0, 117 ± 57.0, and 275 ± 67.0 cells/mm2 for grades 0, 0.5 + , 1 + , 2 + , 3 + , and 4 + , respectively. For the same clinical categories, the average manual cell counts were 6 ± 4.0, 18 ± 3.0, 34 ± 14.0, 72 ± 32.0, 92 ± 43.0, and 168 ± 65.0 cells/mm2, respectively. Zero cells were detected in the AC of healthy eyes. The automated and manual methods were highly correlated (R = 0.98, p < 0.001) and showed good correlation with the clinical grading (R = 0.88, p < 0.001). A mean AC particle size of 117.4 ± 108.8 µm was obtained by the automated method. CONCLUSIONS: Quantification of the AC cells imaged by SS AS-OCT shows good correlation with categorical clinical severity assessments in uveitis eyes. This approach may provide a more objective method for monitoring uveitis and response to uveitis therapy.

Evaluation of the corneal epithelium in non-Sjögren's and Sjögren's dry eyes: an in vivo confocal microscopy study using HRT III RCM.

BACKGROUND: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). METHODS: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. RESULTS: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). CONCLUSIONS: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.

Comparison of Noncontact Specular and Confocal Microscopy for Evaluation of Corneal Endothelium.

PURPOSE: To compare endothelial cell analysis obtained by noncontact specular and confocal microscopy, using the Konan NSP-9900 and Nidek ConfoScan4 systems, respectively. METHODS: Three groups including 70 healthy eyes, 49 eyes with Fuchs endothelial corneal dystrophy (FECD), and 78 eyes with glaucoma were examined with both the Konan NSP-9900 specular microscope and the Nidek ConfocScan4 confocal microscope. Certified graders at the Doheny Image Reading Center compared corneal endothelial images from both instruments side by side to assess image quality. Endothelial cell density (ECD) measurements were calculated and compared using three different modalities: (1) each instrument's fully automated analysis; (2) each instrument's semiautomatic analysis with grader input; and (3) manual grading methods by certified grader. RESULTS: All normal eyes yielded gradable endothelial images, and most but not all glaucomatous eyes yielded images with high enough image quality to allow grading. In addition, in corneas with severe FECD, poor image quality precluded ECD grading by specular microscopy in 20 eyes (40.8%) but in only 4 (8.2%) confocal images from the same eyes. For the gradable images, the ECD values obtained using the manual grading method from either device were comparable with no statistically significant difference (P>0.05) between specular and confocal devices. Machine-generated ECD values were significantly different from manual results, measuring greater in all cases with specular microscopy. Machine-generated ECD values from confocal microscopy also differed significantly from manual determinations, but not in a consistent direction. Semiautomatic methods for both instruments obtained clinically acceptable ECD values. CONCLUSIONS: Automatic machine-generated ECD measurements differed significantly from manual assessments of corneal endothelium by both specular and confocal microscopy, suggesting that automated results should be used with caution. But ECD values derived manually were comparable between the two devices in both normal and glaucomatous eyes, suggesting that manually graded images from the two instruments can be used interchangeably for reliable ECD measurements. Because of a higher proportion of gradable images, confocal microscopy may be superior to specular microscopy for ECD measurements in FECD.

Validity of a new comprehensive pterygia grading scale for use in clinical research and clinical trial.

PURPOSE: To determine the reliability and agreement of a new comprehensive pterygium grading scale for use in clinical research and clinical trials. METHODS: Thirty eyes with pterygia were enrolled in this study. Primary gaze position and lateral gaze position images were taken of each eye with a modified single-lens reflex camera system. Our grading scale includes five parameters: two hyperemia parameters of pterygia on two different gaze position images and three size parameters, quantifying length, width, and area of the cornea encroachment of pterygium, using ImageJ software. All images were graded on the five parameters by two masked, certified reading center graders. Two graders independently graded all the images to determine inter-grader reliability. One grader regraded the images after 3 days to determine intra-grader reliability. Intraclass correlation coefficient (ICC) and inter-rater agreement statistic (κ) calculations were performed. RESULTS: The intra-grader reliability for hyperemia grading was high on both primary and lateral gazing positions (κ value is 0.93 and 0.96). The inter-grader reliability for hyperemia grading was also good (κ value is 0.85 and 0.87). The mean value of width, length, and area of the cornea encroachment of pterygium was 4.31 ± 2.04 mm, 2.08 ± 1.43 mm, and 7.84 ± 7.62 mm2, respectively. The intra-grader agreement on width, length, and area were excellent, with ICCs of 0.98 (95% CI 0.96-0.99), 0.99 (95% CI 0.98-1.0), and 0.97 (95% CI 0.94-0.99), respectively. The inter-grader agreement on width, length, and area were also excellent, with ICCs of 0.96 (95% CI 0.90-0.98), 0.99 (95% CI 0.98-0.99), and 0.99 (95% CI 0.97-0.99), respectively. CONCLUSIONS: There was excellent intra- and inter-observer reproducibility with the new comprehensive grading scale. This scale could lead to the development of standardized grading assessments and quantification of pterygia that would be valid in clinical research and clinical trials.

Correlation between corneal innervation and inflammation evaluated with confocal microscopy and symptomatology in patients with dry eye syndromes: a preliminary study.

PURPOSE: To evaluate corneal innervation and inflammatory cell infiltration using in vivo confocal microscopy (IVCM) and to correlate these findings with subjective symptoms of dry eye, as measured by the Ocular Surface Disease Index (OSDI) in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE). METHODS: Central corneal images were prospectively captured from 10 age-matched healthy control eyes, 24 eyes with clinically diagnosed NSDE and 44 eyes with clinically diagnosed SSDE, using IVCM (HRT III RCM). Density, tortuosity and reflectivity of corneal nerves, presence of inflammatory dendritic cells (DCs) and OSDI scores were evaluated. RESULTS: Images obtained by IVCM from 78 eyes were analyzed. The density of nerve fibers was 1562 ± 996 µm/frame in the SSDE group, 2150 ± 1015 µm/frame in the NSDE group and 2725 ± 687 µm/frame in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of nerve fibers was decreased in the SSDE (P < 0.001) and the NSDE groups (P = 0.06), with increased nerve tortuosity and decreased reflectivity in both groups (both P < 0.05). The density of DCs was 71.65 ± 72.54 cells/mm2 in the SSDE group, 40.33 ± 31.63 cells/mm2 in the NSDE group and 27.53 ± 5.58 cells/mm2 in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of DCs was increased in the SSDE (P < 0.001) and the NSDE groups (P = 0.07). Significant correlations were found between the nerve density and DC density (r = -0.57, P < 0.001), between the nerve density and OSDI scores (r = -0.91, P < 0.001) and between the nerve reflectivity and OSDI scores (r = -0.75, P < 0.001). CONCLUSIONS: The corneas of eyes affected with NSDE and SSDE are characterized by alterations in corneal innervation and infiltration of inflammatory DCs. Corneal nerve density and reflectivity are correlated with severity of subjective dry eye symptoms, as measured by OSDI score.

Corneal features in ocular graft-versus-host disease by in vivo confocal microscopy.

PURPOSE: The purpose of our study was to determine the morphological features of the corneal epithelial layers, sub-basal nerve plexus and anterior stroma in patients with ocular graft-versus-host disease (oGVHD) compared to non-GVHD dry eyes and normal controls, using in vivo confocal microscopy (IVCM). METHODS: IVCM was used to capture central cornea images from eight volunteers with normal healthy eyes, ten patients with non-GVHD dry eye syndrome (DES) and 15 patients with clinically diagnosed oGVHD, in a cross-sectional study. Morphological changes of the corneal epithelial layers and anterior stroma, characteristics of corneal nerves and presence of dendritic cells (DCs) were then evaluated. RESULTS: IVCM images obtained from 66 eyes were analyzed. The density of superficial epithelial cells was 636.07 ± 101.05 cells/mm2 in the oGVHD group, 827 ± 99.62 cells/mm2 in the DES group and 1277.2 ± 121.42 cells/mm2 in the control group (P < 0.001). The density of wing cells was 4499.79 ± 976.36 cells/mm2 in the oGVHD group, 4662.85 ± 319.72 cells/mm2 in DES group and 6556.38 ± 503.99 cells/mm2 in the control group (p < 0.001). The density of basal cells was 7850.93 ± 723.51 cells/mm2 in the oGVHD group, 8570 ± 913.32 cells/mm2 in DES group and 9759.8 ± 251.99 cells/mm2 in the control group (p < 0.01). The density of nerve fibers was 11.22 ± 5.46 mm/mm2 in the oGVHD group, 14.50 ± 4.27 mm/mm2 in DES group and 19.56 ± 4.75 mm/mm2 in the control group (p < 0.01). The DC density was 67.88 ± 71.82 cells/mm2 in the oGVHD group, 40.06 ± 31.95 cells/mm2 in the DES group and 29.45 ± 8.1 cells/mm2 in the control group (P > 0.05). Visible networks of activated keratocytes were seen in the anterior stroma of eyes with oGVHD and DES, but not in normal controls. CONCLUSIONS: IVCM revealed distinct microstructural changes in the corneas of patients with oGVHD and DES, similar between the two groups. Our findings suggest implications for use of IVCM to evaluate and monitor patients with dry eyes associated or not with GVHD.

Stacking S5-n and f5-n to overcome sterility in indica-japonica hybrid rice.

KEY MESSAGE: Pyramiding of S5 - n and f5 - n cumulatively improved seed-setting rate of indica-japonica hybrids, which provided an effective approach for utilization of inter-subspecific heterosis in rice breeding. Breeding for indica-japonica hybrid rice is an attractive approach to increase rice yield. However, hybrid sterility is a major obstacle in utilization of inter-subspecific heterosis. Wide-compatibility alleles can break the fertility barrier between indica and japonica subspecies, which have the potential to overcome inter-subspecific hybrid sterility. Here, we improved the compatibility of an elite indica restorer line 9311 to a broad spectrum of japonica varieties, by introducing two wide-compatibility alleles, S5-n and f5-n, regulating embryo-sac and pollen fertility, respectively. Through integrated backcross breeding, two near isogenic lines harboring either S5-n or f5-n and a pyramiding line carrying S5-n plus f5-n were obtained, with the recurrent parent genome recovery of 99.95, 99.49, and 99.44 %, respectively. The three lines showed normal fertility when crossed to typical indica testers. When testcrossed to five typical japonica varieties, these lines allowed significant increase of compatibility with constant agronomic performance. The introgressed S5-n could significantly improve 14.7-32.9 % embryo-sac fertility in indica-japonica hybrids. In addition, with the presence of f5-n fragment, S5-n would increase the spikelet fertility from 9.5 to 21.8 %. The introgressed f5-n fragment greatly improved anther dehiscence, embryo-sac and pollen fertility in indica-japonica hybrids, thus leading to improvement of spikelet fertility from 20.4 to 30.9 %. Moreover, the pyramiding line showed 33.6-46.7 % increase of spikelet fertility, suggesting cumulative effect of S5-n and f5-n fragment in seed-set improvement of inter-subspecific hybrids. Our results provided an effective approach for exploiting heterosis between indica and japonica subspecies, which had a profound implication in rice breeding.

ENaC in the Rabbit Lacrimal Gland and its Changes During Sjögren Syndrome and Pregnancy.

PURPOSE: Epithelial sodium channel (ENaC) plays a critical role in the control of Na(+) balance and the development and progression of exocrine gland pathologic condition. The aim of the present study was to investigate the presence of ENaC in the rabbit lacrimal gland (LG) and its potential changes during induced autoimmune dacryoadenitis (IAD) and pregnancy. METHODS: Total messenger RNA (mRNA) of α, ß, and γ subunits was extracted from whole LG, acinar cells, and ductal cells by laser capture microdissection (LCM) for real-time reverse-transcriptase polymerase chain reaction. Lacrimal glands were processed for Western blot and immunofluorescence. RESULTS: Messenger RNA for both α and γ was expressed in whole LG lysates, whereas ß was undetectable. In rabbits with IAD, the levels of mRNA for α and γ were 20.9% and 58.9% lower (P<0.05), whereas no significant changes were observed in term-pregnant rabbits (P=0.152). However, we were unable to detect mRNA of any subunit in LCM specimens of ductal cells because of their low levels. Western blot demonstrated bands for both α (90 kDa) and γ (85 kDa) but ß was undetectable. In rabbits with IAD, densitometry analysis showed that expression of α decreased 22%, whereas γ decreased 26% (P<0.05). In pregnant rabbits, however, α expression was 31% lower, whereas γ expression was 34% lower (P<0.05). From immunofluorescence studies, all subunits were present in ductal cells, whereas virtually no immunoreactivity was detected in acini. No noticeable changes of their distribution pattern and intensity were found in rabbits with IAD or during pregnancy. CONCLUSIONS: The present study demonstrated the presence of ENaC in the rabbit LG and its alterations in IAD and pregnancy, suggesting that ENaC may contribute to the pathogenesis of altered LG secretion and ocular surface symptoms in these animals.

4-{Phen-yl[4-(6-phenyl-2,2'-bipyridin-4-yl)phen-yl]amino}-benzaldehyde.

The title mol-ecule, C35H25N3O, is a tri-phenyl-amine derivative with the 4-position substituted by an aldehyde group, and the 4'-position substituted by a 6-phenyl-2,2'-bi-pyridine group. The whole mol-ecule is non-planar and the dihedral angle between the core benzene and pyridine rings is 36.96 (5)°. The dihedral angle between the phenyl and benzaldehyde groups bonded to the amine N atom is 70.86 (5)°.

OsAP65, a rice aspartic protease, is essential for male fertility and plays a role in pollen germination and pollen tube growth.

Aspartic proteases (APs) comprise a large proteolytic enzyme family widely distributed in animals, microbes, viruses, and plants. The rice genome encodes 96 APs, of which only a few have been functionally characterized. Here, the identification and characterization of a novel AP gene, OsAP65, which plays an indispensable role in pollen tube growth in rice, is reported. The T-DNA insertion line of OsAP65 caused severe segregation distortion. In the progeny derived from an individual heterozygous for the T-DNA insertion, the wild type and T-DNA-carrying heterozygote segregated at a ratio close to 1:1, while homozygotes of disrupted OsAP65 (OsAP65-/-) were not recovered. Reciprocal crosses between heterozygotes and wild-type plants demonstrated that the mutant alleles could not be transmitted through the male gamete. Examination of the anthers from heterozygous plants revealed that the mutant pollen matured normally, but did not germinate or elongate. OsAP65 was expressed in various tissues and the transcript level in heterozygous plants was about half of the amount measured in the wild-type plants. The subcellular localization showed that OsAP65 is a pre-vacuolar compartment (PVC) protein. These results indicated that OsAP65 was essential for rice pollen germination and tube growth.

The role of ethylene in plant temperature stress response.

Temperature influences the seasonal growth and geographical distribution of plants. Heat or cold stress occur when temperatures exceed or fall below the physiological optimum ranges, resulting in detrimental and irreversible damage to plant growth, development, and yield. Ethylene is a gaseous phytohormone with an important role in plant development and multiple stress responses. Recent studies have shown that, in many plant species, both heat and cold stress affect ethylene biosynthesis and signaling pathways. In this review, we summarize recent advances in understanding the role of ethylene in plant temperature stress responses and its crosstalk with other phytohormones. We also discuss potential strategies and knowledge gaps that need to be adopted and filled to develop temperature stress-tolerant crops by optimizing ethylene response.

Comparative transcriptomics provides novel insights into the mechanisms of selenium accumulation and transportation in tea cultivars (Camellia sinensis (L.) O. Kuntze).

Tea plants (Camellia sinensis) show discrepancies in selenium accumulation and transportation, the molecular mechanisms of which are not well understood. Hence, we aimed to conduct a systematic investigation of selenium accumulation and transportation mechanisms in different tea cultivars via transcriptome analysis. The Na2SeO3 and Na2SeO4 treatments improved selenium contents in the roots and leaves of three tea cultivars. The high selenium-enrichment ability (HSe) tea cultivars accumulated higher selenium contents in the leaves than did the low selenium-enrichment ability (LSe) tea cultivars. Transcriptome analysis revealed that differentially expressed genes (DEGs) under the Na2SeO3 and Na2SeO4 treatments were enriched in flavonoid biosynthesis in leaves. DEGs under the Na2SeO3 treatment were enriched in glutathione metabolism in the HSe tea cultivar roots compared to those of the LSe tea cultivar. More transporters and transcription factors involved in improving selenium accumulation and transportation were identified in the HSe tea cultivars under the Na2SeO3 treatment than in the Na2SeO4 treatment. In the HSe tea cultivar roots, the expression of sulfate transporter 1;2 (SULTR1;2) and SULTR3;4 increased in response to Na2SeO4 exposure. In contrast, ATP-binding cassette transporter genes (ABCs), glutathione S-transferase genes (GSTs), phosphate transporter 1;3 (PHT1;3), nitrate transporter 1 (NRT1), and 34 transcription factors were upregulated in the presence of Na2SeO3. In the HSe tea cultivar leaves, ATP-binding cassette subfamily B member 11 (ABCB11) and 14 transcription factors were upregulated under the Na2SeO3 treatment. Among them, WRKY75 was explored as a potential transcription factor that regulated the accumulation of Na2SeO3 in the roots of HSe tea cultivars. This study preliminary clarified the mechanism of selenium accumulation and transportation in tea cultivars, and the findings have important theoretical significance for the breeding and cultivation of selenium-enriched tea cultivars.

Two quinoline-based two-photon fluorescent probes for imaging of viscosity in subcellular organelles of living HeLa cells.

Two viscosity-sensitive two-photon fluorescent probes (QL and QLS) were designed and synthesized, which can be localized in lysosome and mitochondria in living HeLa cells, respectively. As the increases of viscosity from 2.55 to 1150 cP, the fluorescence quantum yield (Φ) of QL and QLS was increased by 28-fold and 37-fold, respectively. At the same time, its effective two-photon absorption cross section (ΦδTPA) was enhanced by 15-fold and 16-fold, respectively. Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with QL and QLS, revealed that lysosomal viscosity ranged from 100.76 to 254.74 cP and mitochondrial viscosity ranged from 92.21 to 286.79 cP. This type of fluorescent probe is helpful in the design and application of materials for monitoring diseases associated with abnormal viscosity.

Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

PURPOSE: To test the hypothesis that expression of Na(+)/K(+)-ATPase subunits in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) changes. METHODS: LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. The LGs were processed for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence for the detection of mRNA and proteins of the α1, α2, ß1, ß2, and ß3 subunits of Na(+)/K(+)-ATPase. RESULTS: In the rabbits with IAD, mRNA levels of α1, ß1, and ß3 from whole LGs were significantly lower. In samples of acini and epithelial cells from various duct segments, collected by LCM, mRNA levels of α1, ß1, ß2, and ß3 were significantly lower in the rabbits with IAD, although mRNA for α2 could not be detected. However, western blots demonstrated that all five subunits were significantly higher in the rabbits with IAD, although their distribution patterns were similar to those of the control rabbits, as demonstrated by immunofluorescence. CONCLUSIONS: The data presented herein demonstrated significant changes in mRNA and protein expressions of Na(+)/K(+)-ATPase subunits in rabbits with IAD, suggesting that these changes may play a role in the pathogenesis of Sjögren's syndrome and altered LG secretion, as observed in these animals.