Publications by authors named "Yingzhen Yang"

33 Publications

Thermal-responsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds.

Hortic Res 2020 Aug 1;7(1):114. Epub 2020 Aug 1.

USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV, 25430, USA.

The Dormancy-associated MADS-box (DAM) gene cluster in peach serves as a key regulatory hub on which the seasonal temperatures act and orchestrate dormancy onset and exit, chilling response and floral bud developmental pace. Yet, how different temperature regimes interact with and regulate the six linked DAM genes remains unclear. Here, we demonstrate that chilling downregulates DAM1 and DAM3-6 in dormant floral buds with distinct patterns and identify DAM4 as the most abundantly expressed one. We reveal multiple epigenetic events, with tri-methyl histone H3 lysine 27 (H3K27me3) induced by chilling specifically in DAM1 and DAM5, a 21-nt sRNA in DAM3 and a ncRNA induced in DAM4. Such induction is inversely correlated with downregulation of their cognate DAMs. We also show that the six DAMs were hypermethylated, associating with the production of 24-nt sRNAs. Hence, the chilling-responsive dynamic of the different epigenetic elements and their interactions likely define distinct expression abundance and downregulation pattern of each DAM. We further show that the expression of the five DAMs remains steadily unchanged or continuously downregulated at the ensuing warm temperature after chilling, and this state of regulation correlates with robust increase of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in DAM4. Such robust increase of repressive epigenetic marks may irreversibly reinforce the chilling-imposed repression of DAMs to ensure flower-developmental programming free from any residual DAM inhibition. Taken together, we reveal novel information about genetic and epigenetic regulation of the DAM cluster in peach, which will be of fundamental significance in understanding of the regulatory mechanisms underlying chilling requirement and dormancy release, and of practical application for improvement of plasticity of flower time and bud break in fruit trees to adapt changing climates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41438-020-0336-yDOI Listing
August 2020

A key 'foxy' aroma gene is regulated by homology-induced promoter indels in the iconic juice grape 'Concord'.

Hortic Res 2020 Apr 18;7(1):67. Epub 2020 Apr 18.

US Department of Agriculture-Agricultural Research Service, Grape Genetics Research Unit, Geneva, NY, USA.

'Concord', the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special 'foxy' aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key 'foxy' aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in 'Concord' and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the 'foxy' aroma trait and makes it genetically trackable and amenable in grapevine breeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41438-020-0304-6DOI Listing
April 2020

The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine.

Plant J 2021 03 21;105(6):1495-1506. Epub 2021 Jan 21.

Beijing Key Laboratory of Grape Science and Enology, and CAS Key Laboratory of Plant Resources, Institute of Botany, Innovation Academy for Seed Design, the Chinese Academy of Science, Beijing, 100093, China.

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tpj.15127DOI Listing
March 2021

Thermal-responsive genetic and epigenetic regulation of cluster controlling dormancy and chilling requirement in peach floral buds.

Hortic Res 2020 1;7:114. Epub 2020 Aug 1.

USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430 USA.

The () gene cluster in peach serves as a key regulatory hub on which the seasonal temperatures act and orchestrate dormancy onset and exit, chilling response and floral bud developmental pace. Yet, how different temperature regimes interact with and regulate the six linked genes remains unclear. Here, we demonstrate that chilling downregulates in dormant floral buds with distinct patterns and identify as the most abundantly expressed one. We reveal multiple epigenetic events, with tri-methyl histone H3 lysine 27 (H3K27me3) induced by chilling specifically in and , a 21-nt sRNA in and a ncRNA induced in 4. Such induction is inversely correlated with downregulation of their cognate s. We also show that the six s were hypermethylated, associating with the production of 24-nt sRNAs. Hence, the chilling-responsive dynamic of the different epigenetic elements and their interactions likely define distinct expression abundance and downregulation pattern of each . We further show that the expression of the five s remains steadily unchanged or continuously downregulated at the ensuing warm temperature after chilling, and this state of regulation correlates with robust increase of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in . Such robust increase of repressive epigenetic marks may irreversibly reinforce the chilling-imposed repression of s to ensure flower-developmental programming free from any residual inhibition. Taken together, we reveal novel information about genetic and epigenetic regulation of the cluster in peach, which will be of fundamental significance in understanding of the regulatory mechanisms underlying chilling requirement and dormancy release, and of practical application for improvement of plasticity of flower time and bud break in fruit trees to adapt changing climates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41438-020-0336-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395172PMC
August 2020

A key 'foxy' aroma gene is regulated by homology-induced promoter indels in the iconic juice grape 'Concord'.

Hortic Res 2020 18;7:67. Epub 2020 Apr 18.

1US Department of Agriculture-Agricultural Research Service, Grape Genetics Research Unit, Geneva, NY USA.

'Concord', the most well-known juice grape with a parentage of the North American grape species L., possesses a special 'foxy' aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species . Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key 'foxy' aroma gene, anthraniloyl-CoA:methanol acyltransferase (), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in promoters highly associated with high levels of expression and MA accumulation in 'Concord' and other -derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 germplasm. Moreover, functional impact of these indels was validated in transgenic . Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the locus in some grapes. Elucidation of the genetic regulation advances our understanding of the 'foxy' aroma trait and makes it genetically trackable and amenable in grapevine breeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41438-020-0304-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166211PMC
April 2020

RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines.

BMC Plant Biol 2019 Feb 18;19(1):80. Epub 2019 Feb 18.

USDA-ARS Grape Genetics Research Unit, Geneva, NY, 14456, USA.

Background: Gibberellins (GAs) and their regulator DELLA are involved in many aspects of plant growth and development and most of our current knowledge in the DELLA-facilitated GA signaling was obtained from the studies of annual species. To understand GA-DELLA signaling in perennial species, we created ten GA-insensitive transgenic grapevines carrying a DELLA mutant allele (Vvgai1) in the background of Vitis vinifera 'Thompson Seedless' and conducted comprehensive analysis of their RNA expression profiles in the shoot, leaf and root tissues.

Results: The transgenic lines showed varying degrees of dwarf stature and other typical DELLA mutant phenotypes tightly correlated with the levels of Vvgai1 expression. A large number of differentially expressed genes (DEGs) were identified in the shoot, leaf and root tissues of the transgenic lines and these DEGs were involved in diverse biological processes; many of the DEGs showed strong tissue specificity and about 30% them carried a DELLA motif. We further discovered unexpected expression patterns of several key flowering induction genes VvCO, VvCOL1 and VvTFL1.

Conclusions: Our results not only confirmed many previous DELLA study findings in annual species, but also revealed new DELLA targets and responses in grapevine, including the roles of homeodomain transcription factors as potential co-regulators with DELLA in controlling the development of grapevine which uniquely possess both vegetative and reproductive meristems at the same time. The contrasting responses of some key flowering induction pathway genes provides new insights into the divergence of GA-DELLA regulations between annual and perennial species in GA-DELLA signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-019-1675-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379989PMC
February 2019

Endothelial‑to‑mesenchymal transition in human idiopathic dilated cardiomyopathy.

Mol Med Rep 2018 Jan 8;17(1):961-969. Epub 2017 Nov 8.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, P.R. China.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and cardiac fibrosis. Emerging evidence indicated that endothelial‑to‑mesenchymal transition (Endo‑MT) is a crucial event during organ fibrosis. This study was performed to clarify whether Endo‑MT contributed to the progression of cardiac fibrosis in DCM. Cardiac samples from patients with DCM and control were obtained. The presence of endothelial markers, cluster of differentiation (CD)31 and vascular endothelial (VE)‑cadherin, and mesenchymal markers, α smooth muscle actin (SMA) and fibroblast‑specific protein 1 (FSP1) was performed using immunohistochemistry. Co‑localization of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Serum procollagen type I carboxy‑terminal propeptide (PICP) and procollagen type III amino‑terminal propeptide (PIIINP) were measured by ELISA. Protein levels of Wnt, β‑catenin and Snail were determined using western blot analysis. Immunohistochemistry and double‑immunofluorescence staining demonstrated that the expression of CD31 and VE‑cadherin were significantly decreased in DCM samples, whereas the FSP‑1, and αSMA were significantly increased. CD31 and VE‑cadherin labeling indexes were respectively negatively correlated with left ventricular end‑diastolic diameter (LVEDD) (CD31 r=‑0.82, P<0.01; VE‑cadherin r=-0.73, P<0.01), while FSP‑1 and αSMA were positively associated with LVEDD (αSMA r=0.65, P<0.01, FSP1 r=0.53, P<0.01) and left ventricular ejection fraction (αSMA r=‑0.18, P<0.05; FSP1 r=‑0.21, P<0.05). Furthermore, PICP and PIIINP levels were positively associated with the co‑expression labeling indexes (CD31/SMA co‑labeling index and PICP r=0.727, P<0.01; CD31/SMA co‑labeling index and PIIINP r=0.741, P<0.01; VE‑Cadherin/FSP‑1 co‑labeling index and PICP r=0.716, P<0.01; VE‑cadherin/FSP‑1 co‑labeling index and PIIINP r=0.648, P<0.05). Western blot analysis indicated that proteins levels of Wnt signaling and snail were significantly increased in DCM samples. These results suggested that Endo‑MT is potentially implicated in the pathogenesis of myocardial fibrosis and remodeling during the development of DCM, indicating a potential therapeutic target for DCM treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mmr.2017.8013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780177PMC
January 2018

A transcriptome analysis of two grapevine populations segregating for tendril phyllotaxy.

Hortic Res 2017 12;4:17032. Epub 2017 Jul 12.

USDA-Agricultural Research Service, Grape Genetics Research Unit, Geneva, NY 14456, USA.

The shoot structure of cultivated grapevine L. typically exhibits a three-node modular repetitive pattern, two sequential leaf-opposed tendrils followed by a tendril-free node. In this study, we investigated the molecular basis of this pattern by characterizing differentially expressed genes in 10 bulk samples of young tendril tissue from two grapevine populations showing segregation of mutant or wild-type shoot/tendril phyllotaxy. One population was the selfed progeny and the other one, an outcrossed progeny of a hybrid, 'Roger's Red'. We analyzed 13 375 expressed genes and carried out in-depth analyses of 324 of them, which were differentially expressed with a minimum of 1.5-fold changes between the mutant and wild-type bulk samples in both selfed and cross populations. A significant portion of these genes were direct -binding targets of 14 transcription factor families that were themselves differentially expressed. Network-based dependency analysis further revealed that most of the significantly rewired connections among the 10 most connected hub genes involved at least one transcription factor. TCP3 and MYB12, which were known important for plant-form development, were among these transcription factors. More importantly, TCP3 and MYB12 were found in this study to be involved in regulating the lignin gene , which is important to plant-form development. A further support evidence for the roles of TCP3-MYB12-PRX52 in contributing to tendril phyllotaxy was the findings of two other lignin-related genes uniquely expressed in the mutant phyllotaxy background.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/hortres.2017.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506248PMC
July 2017

Messenger RNA exchange between scions and rootstocks in grafted grapevines.

BMC Plant Biol 2015 Oct 19;15:251. Epub 2015 Oct 19.

United States Department of Agriculture, Agricultural Research Service, Grape Genetics Research Unit, Geneva, NY, 14456, USA.

Background: Grafting has been widely practiced for centuries in the propagation and production of many vegetable and fruit species. However, the underlying molecular and genetic mechanisms for how the graft partners interact with each other to produce a successful graft remain largely unknown. We hypothesized that genome-wide mRNA exchanges, which were recently documented in grafted model plant species, are a general phenomenon widely present in grafted plants, including those in vegetable and fruit species, and have specific genotype- and environment-dependent characteristics modulating plant performance.

Methods: Using diagnostic SNPs derived from high throughput genome sequencing, we identified and characterized the patterns of genome-wide mRNA exchanges across graft junctions in grafted grapevines grown in the in vitro and field conditions.

Results: We identified more than 3000 genes transporting mRNAs across graft junctions. These genes were involved in diverse biological processes and those involved in basic cellular, biosynthetic, catabolic, and metabolic activities, as well as responses to stress and signal transduction, were highly enriched. Field-grown mature grafts had much fewer genes transmitting mRNAs than the in vitro young grafts (987 vs. 2679). These mobile mRNAs could move directionally or bi-directionally between scions and rootstocks. The mRNA transmission rates of these genes were generally low, with 65% or more having transmission rates lower than 0.01. Furthermore, genotypes, graft combinations and growth environments had impact on the directions of mRNA movement as well as the numbers and species of mRNAs being exchanged. Moreover, we found evidence for the presences of both passive and selective mechanisms underlying long distance mRNA trafficking in grafted grapevines.

Conclusions: We extended the studies of mRNA exchanges in model species to grapevines and demonstrated that genomic-scale mRNA exchange across graft junctions occurred in grapevines in a passive or genotype and environment-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-015-0626-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612405PMC
October 2015

Learning Super-Resolution Jointly From External and Internal Examples.

IEEE Trans Image Process 2015 Nov 5;24(11):4359-71. Epub 2015 Aug 5.

Single image super-resolution (SR) aims to estimate a high-resolution (HR) image from a low-resolution (LR) input. Image priors are commonly learned to regularize the, otherwise, seriously ill-posed SR problem, either using external LR-HR pairs or internal similar patterns. We propose joint SR to adaptively combine the advantages of both external and internal SR methods. We define two loss functions using sparse coding-based external examples, and epitomic matching based on internal examples, as well as a corresponding adaptive weight to automatically balance their contributions according to their reconstruction errors. Extensive SR results demonstrate the effectiveness of the proposed method over the existing state-of-the-art methods, and is also verified by our subjective evaluation study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TIP.2015.2462113DOI Listing
November 2015

Multiple loss-of-function 5-O-glucosyltransferase alleles revealed in Vitis vinifera, but not in other Vitis species.

Theor Appl Genet 2014 Nov 11;127(11):2433-51. Epub 2014 Sep 11.

USDA-ARS Grape Genetics Research Unit, Geneva, NY, 14456, USA.

Key Message: Wild and loss-of-function alleles of the 5 - O - glucosyltransferase gene responsible for synthesis of diglucoside anthocyanins in Vitis were characterized. The information aids marker development for tracking this gene in grape breeding. Anthocyanins in red grapes are present in two glycosylation states: monoglucoside (3-O-glucoside) and diglucoside (3, 5-di-O-glucoside). While monoglucoside anthocyanins are present in all pigmented grapes, diglucoside anthocyanins are rarely found in the cultivated grape species Vitis vinifera. Biochemically 3-O-glucoside anthocyanins can be converted into 3,5-di-O-glucoside anthocyanins by a 5-O-glucosyltransferase. In this study, we surveyed allelic variation of the 5-O-glucosyltransferase gene (5GT) in 70 V. vinifera ssp. vinifera cultivars, 52 V. vinifera ssp. sylvestris accessions, 23 Vitis hybrid grapes, and 22 accessions of seven other Vitis species. Eighteen 5GT alleles with apparent loss-of-function mutations, including seven premature stop codon mutations and six frameshift indel mutations, were discovered in V. vinifera, but not in the other Vitis species. A total of 36 5GT alleles without apparent loss-of-function mutations (W-type) were identified. These W-type alleles were predominantly present in wild Vitis species, although a few of them were also found in some V. vinifera accessions. We further evaluated some of these 5GT alleles in producing diglucoside anthocyanins by analyzing the content of diglucoside anthocyanins in a set of representative V. vinifera cultivars. Through haplotype network analysis we revealed that V. vinifera ssp. vinifera and its wild progenitor V. vinifera ssp. sylvestris shared many loss-of-function 5GT alleles and extensive divergence of the 5GT alleles was evident within V. vinifera. This work advances our understanding of the genetic diversity of 5GT and provides a molecular basis for future marker-assisted selection for improving this important wine quality trait.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00122-014-2388-6DOI Listing
November 2014

In vivo delivery of adenoviral vector containing interleukin-17 receptor a reduces cardiac remodeling and improves myocardial function in viral myocarditis leading to dilated cardiomyopathy.

PLoS One 2013 20;8(8):e72158. Epub 2013 Aug 20.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China ; Department of Cardiology, Xinhua Hospital affiliated to Shanghai Jiaotong University, Shanghai, China.

Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072158PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748008PMC
April 2014

Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots.

PLoS One 2013 16;8(7):e69463. Epub 2013 Jul 16.

United States Department of Agriculture-Agricultural Research Service, Grape Genetics Research Unit, Geneva, New York, United States of America.

Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5' end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069463PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712915PMC
February 2014

Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3 infection and its potential role in cardiac fibrosis.

Virus Res 2012 Oct 4;169(1):188-94. Epub 2012 Aug 4.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

CVB3 virus tropism and tissue access are modulated by cardiac microvascular endothelial cells (CMVECs) in the context of microvasculature. This study was designed to examine biological behaviors of CMVECs following CVB3 infection and its possible effects on cardiac remodeling. Data demonstrated that CVB3 increased caspase-3 activities, Bax/Bcl-2 protein ratio and TGF-β1 levels in CMVECs, accompanying with elevated microvascular permeability. Double immunofluorescence revealed co-localization of endothelial markers (CD31 and VE-cadherin) and mesenchymal markers (FSP1 and αSMA) in infected CMVECs. Western blot demonstrated that CVB3 significantly decreased the expression of endothelial markers and increased the expression of mesenchymal markers, which were reversed by SB431542 (inhibitor of TGF-β1), indicating that endothelial-to-mesenchymal transition following CVB3 infection was probably induced by CMVECs-derived TGF-β1. Excess extracellular matrix was produced by myocardial cells incubated with supernatants of infected CMVECs. Our results displayed that CVB3 induced notable biological changes of CMVECs, which may contribute to cardiac fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2012.07.027DOI Listing
October 2012

Characterization of polyphenolic metabolites in the seeds of Vitis germplasm.

J Agric Food Chem 2012 Feb 26;60(5):1291-9. Epub 2012 Jan 26.

Department of Horticulture, Cornell University, Ithaca, New York 14853, United States.

The composition and content of polyphenols in the seeds of 91 grape accessions from 17 Vitis species were characterized. Eleven compounds, including 2 gallic derivatives, 3 monomeric flavan-3-ols, 3 flavonols, resveratrol, and procyanidin B1 and B2, were identified via HPLC-MS and quantified by HPLC-DAD. In addition, seventeen dimeric and trimeric flavan-3-ols were also quantified. Tremendous variation was observed both among and within species for these compounds. Monomeric flavan-3-ols were the most abundant polyphenols in seeds, followed by dimeric and trimeric flavan-3-ols, which collectively accounted for more than 96% of the total polyphenols. V. palmata, V. vinifera, and V. vulpina had significantly higher content of total polyphenols than other species. A number of Vitis accessions with high content of various types of seed polyphenols were identified, and they can serve as potential germplasm for improving the composition and content of seed polyphenols in cultivated grapes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jf2046637DOI Listing
February 2012

Blockade of interleukin-17A protects against coxsackievirus B3-induced myocarditis by increasing COX-2/PGE2 production in the heart.

FEMS Immunol Med Microbiol 2012 Apr 22;64(3):343-51. Epub 2011 Dec 22.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China.

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1574-695X.2011.00918.xDOI Listing
April 2012

Characterization of grape Gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis.

Transgenic Res 2012 Aug 29;21(4):725-41. Epub 2011 Oct 29.

Grape Genetics Research Unit, USDA-ARS, 630W North Street, Geneva, NY 14456, USA.

We generated 12 different mutations in the grape Gibberellin Insensitive1 (VvGAI1) sequences, transformed them into Arabidopsis under the control of 35S, Arabidopsis GAI or grape GAI1 promoter, and evaluated the impact of these mutant alleles on plant growth and development. These VvGAI1 sequence variants included some mimics of the known GAI-like mutant alleles discovered in grape, wheat, barley, corn, Brassica, and Arabidopsis. In general, plant height and related traits such as length of internodes and inflorescences were significantly reduced for most of the mutant alleles studied, regardless of which promoter was used. Interestingly, the numbers of rosette leaves and lateral branches were generally reduced when a 35S promoter was used to express the mutant alleles, but increased when an Arabidopsis or grape GAI promoter was used. Furthermore, the 35S plants often displayed curly and small leaves. In contrast, the leaves of the plants carrying mutant alleles controlled by a GAI promoter were of variable size, dark green and rarely curly. In addition, when certain VvGAI1 mutant alleles were under the control of the grape GAI1 promoter, the number of pods on inflorescences was significantly increased, but some of the pods produced few seeds due to partial sterility. On the basis of the systematic evaluation of various VvGAI1 mutant alleles in Arabidopsis, we concluded that the VvGAI1 mutant alleles mimicking the GAI or GAI-like mutant variants discovered in wheat, barley and Brassica could potentially be useful for the improvement of grapevine plant architecture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-011-9565-zDOI Listing
August 2012

The role of Th17 cells and regulatory T cells in Coxsackievirus B3-induced myocarditis.

Virology 2011 Dec 11;421(1):78-84. Epub 2011 Oct 11.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

IL-17-producing (Th17) and regulatory T (Treg) cells have been well established in the pathogenesis of many inflammatory diseases. To assess whether Th17 and Treg were altered in acute virus-induced myocarditis (AVMC) mice, we assessed Th17/Treg functions on different levels in AVMC. It was shown that the expression of splenic Th17 cells and Th17-related cytokines (IL-17A, IL-21) markedly increased. Interestingly, the expression of splenic Treg cells and Treg-related cytokines (TGF-β, IL-10) also significantly increased. Using neutralization of IL-17 in the AVMC, we found that Treg cells roughly decreased compared with isotype control mice. However, T cells and perforin dramatically increased, followed by a marked reduction in CVB3 replication. The results suggested that Th17 cells possibly contributed to viral replication through the action of Treg cells in mediating T cells and perforin response in AVMC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2011.09.006DOI Listing
December 2011

Astragaloside IV attenuates myocardial fibrosis by inhibiting TGF-β1 signaling in coxsackievirus B3-induced cardiomyopathy.

Eur J Pharmacol 2011 May 1;658(2-3):168-74. Epub 2011 Mar 1.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

Myocardial fibrosis plays an important role in coxsackievirus B3 (CVB3) induced dilated cardiomyopathy. Excessive transforming growth factor (TGF)-β1 contributes to a pathologic excess of tissue fibrosis. We investigated the effect of astragaloside IV on myocardial fibrosis in CVB3-induced dilated cardiomyopathy. BALB/c mice were inoculated with CVB3 to induce acute viral myocarditis on day 7 (acute VMC group), monthly for 3 months to induce chronic myocarditis (chronic VMC group), and monthly for 9 months to induce dilated cardiomyopathy (DCM group). The same method was used for the DCM+Astra group as that of the DCM group, but former group was given with astragaloside IV-containing drinking water. Compared to DCM group, astragaloside IV treatment significantly increased the survival rate. Histological findings and the collagen volume fraction showed that astragaloside IV decreased fibrosis in heart tissues. Astragaloside IV decreased the level of the serum carboxy-terminal propeptide of procollagen type I (PICP) and the ratio of PICP/ N-terminal type I procollagen propeptide (PINP). Ameliorated myocardial fibrosis was consistent with the downregulated expression of TGF-β1 and its downstream pSmad2/3 and Smad4 in the myocardium of the DCM+Astra group compared to the DCM group. The level of type I collagen was lower in the DCM+Astra group than the DCM group. The same effect was found in the in vitro study. These findings showed that astragaloside IV had a potent preventive effect on myocardial fibrosis in CVB3-induced dilated cardiomyopathy that might be due to downregulation of TGF-β1-Smad signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejphar.2011.02.040DOI Listing
May 2011

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2.

Front Physiol 2010 22;1:24. Epub 2010 Sep 22.

Department of Plant Biology, Carnegie Institution for Science Stanford, CA, USA.

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r(2) = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2010.00024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059934PMC
May 2011

Coxsackievirus B3 infection promotes generation of myeloid dendritic cells from bone marrow and accumulation in the myocardium.

Int Immunopharmacol 2009 Oct 4;9(11):1304-12. Epub 2009 Aug 4.

Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

Myocarditis is associated with increased number of CD4+ T cells in the myocardium after coxsackievirus B3 (CVB3) infection. Previous studies show that CD11c+ myeloid dendritic cells (mDC) loaded with myosin could induce myocarditis. This study aims to investigate the generation and accumulation of mDC in CVB3-induced myocarditis. The presence of mDC in myocardium was detected by immunohistochemisty. Bone marrow-derived mDC were generated from uninfected and CVB3-infected mice. The percentage of CD11c+ mDC on cultured cells and mean fluorescence index (MFI) of double positive cells (CD11c+CD40+, CD11c+CD80+) were measured by flow cytometry. The expression of chemokine receptors (CCR5, CCR7) on mDC and chemokines (CCL4, CCL19) in the myocardium was detected. The migration of mDC in response to CCL4 or CCL19 was measured by chemotaxis assay. Mature mDC were elevated in the myocardium from CVB3-infected mice. The percentage of mDC generated from CVB3-infected group was increased. The MFI of CD11c+CD40+ and CD11c+CD80+ was increased in CVB3-infected group. The mDC showed a down-regulation of CCR5 and unaffected CCR7 mRAN levels associated with elevated CCL4 and CCL19 in the myocardium in CVB3-infected group. Numbers of migrating bone marrow-derived mDC from CVB3-infected mice were increased in vitro. We conclude that CVB3 infection induced the greater generation of mDC from bone marrow and accumulation of mature mDC in myocardial tissues. This migration was associated with increased levels of both CCL4 and CCL19 in the heart tissue. These suggest that blocking the migration of mDC may provide a novel therapy for myocarditis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.intimp.2009.07.014DOI Listing
October 2009

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K channels in Arabidopsis guard cells.

Plant J 2009 Jul 19;59(2):207-20. Epub 2009 Mar 19.

Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA.

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-313X.2009.03872.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2827207PMC
July 2009

Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool.

Plant Methods 2008 Feb 19;4. Epub 2008 Feb 19.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0116, USA.

Background: A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.

Results: A promoter, pGC1(At1g22690), drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase) and yellow cameleon YC3.60 (GFP-based calcium FRET reporter). Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca2+ dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The GC1 promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant too-many-mouths (tmm). Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using pGC1 was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression.

Conclusion: The pGC1 promoter described here drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression or gene silencing. It is also applicable to reduce specific gene expression in guard cells, providing a method for circumvention of limitations arising from genetic redundancy and lethality. These advances could be very useful for manipulating signaling pathways in guard cells and modifying plant performance under stress conditions. In addition, new guard cell and mesophyll cell-specific 23,000 gene microarray data are made publicly available here.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1746-4811-4-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323621PMC
February 2008

Conserved C-terminal motifs of the Arabidopsis proteins APETALA3 and PISTILLATA are dispensable for floral organ identity function.

Plant Physiol 2007 Dec 26;145(4):1495-505. Epub 2007 Oct 26.

Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755.

The B-class genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis (Arabidopsis thaliana) and their orthologs in other species have been the focus of studies to elucidate the development of petals and stamens in angiosperm flowers. Evolutionary analysis indicates that B-class genes have undergone multiple gene duplication events in angiosperms. The resultant B-class lineages are characterized by short, conserved amino acid sequences at the extreme C-terminal end of the B-class proteins. AP3 is a member of the euAP3 lineage that contains both the euAP3 and PI-derived motifs at the C terminus. PI is a member of the PI lineage that contains the C-terminal PI motif at the C terminus. Despite conservation over a wide evolutionary distance, the function of C-terminal motifs is not well understood. In this study, we demonstrate that truncated forms of AP3 and PI, which lack the conserved C-terminal motifs, function to direct floral organ identity specification in Arabidopsis plants. By contrast, larger truncations, which remove the third putative amphipathic alpha-helix in the K domain of AP3 or PI, are nonfunctional. We conclude that the euAP3 and PI-derived motifs of AP3 and the PI motif of PI are not essential for floral organ identity function of AP3 and PI in Arabidopsis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.107.105346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151695PMC
December 2007

KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells.

Plant Mol Biol 2008 Jan 23;66(1-2):61-72. Epub 2007 Oct 23.

Istituto di Biofisica-CNR, Via De Marini 6, Genova 16149, Italy.

The Shaker potassium channels are tetrameric proteins formed by the assembly of four alpha-subunits. The oligomerization can occur among both homo- and hetero-alpha-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1::GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1::GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1::GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1::GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1::GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11103-007-9252-xDOI Listing
January 2008

DORNROSCHEN-LIKE, an AP2 gene, is necessary for stamen emergence in Arabidopsis.

Plant Mol Biol 2007 Oct 8;65(3):219-32. Epub 2007 Aug 8.

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.

To identify novel genes in petal and stamen development, a genetic screen was carried out for enhancers of the unusual B class mutant pistillata-5 (pi-5). In pi-5 flowers, second whorl organs develop as sepals rather than petals, but third whorl stamens are normal. One pi-5 enhancer, dornröschen-like-2 (drnl-2), results in third whorl positions developing as filamentous organs. In addition to enhancing the pi-5 phenotype, drnl-2 mutants also exhibit a phenotype in a wild-type PI background. Although stamen primordia are morphologically visible during early stages of flower development, they fail to enlarge in drnl-2 mutants. DRNL, which encodes a single AP2 domain protein, is expressed in a dynamic pattern in the embryo, seedling, and flower. Analysis of both the drnl-2 mutant phenotype and the DRNL expression pattern in flowers suggests that DRNL plays a critical role in stamen emergence in Arabidopsis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11103-007-9210-7DOI Listing
October 2007

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure.

PLoS Biol 2006 Oct;4(10):e327

Cell and Developmental Biology Section, Division of Biological Sciences and Center for Molecular Genetics, University of California San Diego, La Jolla, California, USA.

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.0040327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592316PMC
October 2006

Enhanced myocardial cathepsin B expression in patients with dilated cardiomyopathy.

Eur J Heart Fail 2006 May 15;8(3):284-9. Epub 2006 Feb 15.

Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Fenglin Road 180, Shanghai 200032, PR China.

Objective: Cathepsin B is a prominent lysosomal protease and is involved in apoptosis as well as degradation of myofibrillar proteins in myocardial infarction. The aim of this study was to investigate myocardial cathepsin B expression in failing and non-failing human hearts.

Methods: Tissue samples were taken from transplanted left ventricles from 20 patients with dilated cardiomyopathy and 5 non-failing donor hearts that could not be transplanted for technical reasons. Myocardial cathepsin B expression was determined by immunohistochemistry, the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Apoptosis was assessed by TUNEL staining.

Results: Positive cathepsin B staining was found in failing and non-failing hearts. The expression of cathepsin B at mRNA and protein levels was significantly higher in failing hearts compared with non-failing hearts. Correlation analysis revealed that cathepsin B at mRNA and protein levels negatively correlated with EF (r=0.66, p=0.002 and r=0.492, p=0.028, respectively) in patients with heart failure. The apoptotic index was 0.015+/-0.006 in failing hearts and 0.002+/-0.001 in non-failing hearts (p<0.01).

Conclusion: Increased myocardial expression of cathepsin B was found in patients with heart failure suggesting that cathepsin B might play a role in the genesis and development of heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejheart.2005.09.004DOI Listing
May 2006

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats.

Life Sci 2006 Mar 23;78(17):1998-2005. Epub 2005 Nov 23.

Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, No. 555 Zuchongzhi Road, Zhangjiang Pudong, Shanghai, China.

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lfs.2005.09.034DOI Listing
March 2006

Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins.

Plant Mol Biol 2004 May;55(1):45-59

Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.

The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11103-004-0416-7DOI Listing
May 2004
-->