Saussurea involucrata (Snow Lotus) ICE1 and ICE2 Orthologues Involved in Regulating Cold Stress Tolerance in Transgenic Arabidopsis.

As with other environmental stresses, cold stress limits plant growth, geographical distribution, and agricultural productivity. CBF/DREB (CRT-binding factors/DRE-binding proteins) regulate tolerance to cold/freezing stress across plant species. ICE (inducer of CBF expression) is regarded as the upstream inducer of CBF expression and plays a crucial role as a main regulator of cold acclimation. Snow lotus (Saussurea involucrata) is a well-known traditional Chinese herb. This herb is known to have greater tolerance to cold/freezing stress compared to other plants. According to transcriptome datasets, two putative ICE homologous genes, SiICE1 and SiICE2, were identified in snow lotus. The predicted SiICE1 cDNA contains an ORF of 1506 bp, encoding a protein of 501 amino acids, whereas SiICE2 cDNA has an ORF of 1482 bp, coding for a protein of 493 amino acids. Sequence alignment and structure analysis show SiICE1 and SiICE2 possess a S-rich motif at the N-terminal region, while the conserved ZIP-bHLH domain and ACT domain are at the C-terminus. Both SiICE1 and SiICE2 transcripts were cold-inducible. Subcellular localization and yeast one-hybrid assays revealed that SiICE1 and SiICE2 are transcriptional regulators. Overexpression of SiICE1 (35S::SiICE1) and SiICE2 (35S::SiICE2) in transgenic Arabidopsis increased the cold tolerance. In addition, the expression patterns of downstream stress-related genes, CBF1, CBF2, CBF3, COR15A, COR47, and KIN1, were up-regulated when compared to the wild type. These results thus provide evidence that SiICE1 and SiICE2 function in cold acclimation and this cold/freezing tolerance may be regulated through a CBF-controlling pathway.

The Direct Involvement of Dark-Induced Tic55 Protein in Chlorophyll Catabolism and Its Indirect Role in the MYB108-NAC Signaling Pathway during Leaf Senescence in Arabidopsis thaliana.

The chloroplast relies on proteins encoded in the nucleus, synthesized in the cytosol and subsequently transported into chloroplast through the protein complexes Toc and Tic (Translocon at the outer/inner membrane of chloroplasts). A Tic complex member, Tic55, contains a redox-related motif essential for protein import into chloroplasts in peas. However, Tic55 is not crucial for protein import in Arabidopsis. Here, a tic55-II-knockout mutant of Arabidopsis thaliana was characterized for Tic55 localization, its relationship with other translocon proteins, and its association with plant leaf senescence when compared to the wild type. Individually darkened leaves (IDLs) obtained through dark-induced leaf senescence were used to demonstrate chlorophyll breakdown and its relationship with plant senescence in the tic55-II-knockout mutant. The IDLs of the tic55-II-knockout mutant contained higher chlorophyll concentrations than those of the wild type. Our microarray analysis of IDLs during leaf senescence identified seven senescence-associated genes (SAGs) that were downregulated in the tic55-II-knockout mutant: ASP3, APG7, DIN2, DIN11, SAG12, SAG13, and YLS9. Real-time quantitative PCR confirmed the reliability of microarray analysis by showing the same expression patterns with those of the microarray data. Thus, Tic55 functions in dark-induced aging in A. thaliana by indirectly regulating downstream SAGs expression. In addition, the expression of four NAC genes, including ANAC003, ANAC010, ANAC042, and ANAC075 of IDL treated tic55-II-knockout mutant appeared to be downregulated. Yeast one hybrid assay revealed that only ANAC003 promoter region can be bound by MYB108, suggesting that a MYB-NAC regulatory network is involved in dark-stressed senescence.

Overexpression of Lilium formosanumMADS-box (LFMADS) Causing Floral Defects While Promoting Flowering in Arabidopsis thaliana, Whereas Only Affecting Floral Transition Time in Nicotiana tabacum.

The Formosa lily (Lilium formosanum) is one of the most common horticultural species in Taiwan. To explore gene regulation involved in this species, we used transcriptome analysis to generate PH-FB (mixed floral buds) and PH-LF (mature leaves) datasets. Combination of the PH-FB and PH-LF constructed a de novo assembly of the ALL dataset, including 18,041 contigs and 23,807 unigenes by Nr, GO, COG, and KEGG databases. The differential gene expression (DGE) analysis revealed 9937 genes were upregulated while 10,383 genes were downregulated in the developing floral buds compared to mature leaves. Seven putative genes (LFMADS1 to 7) encoding floral organ identity proteins were selected for further analysis. LFMADS1-6 genes were specifically expressed in the floral organ, while LFMADS7 in the floral buds and mature leaves. Phylogenetic analysis revealed that LFMADS1-3 is classified into B-class, LFMADS4 into C-class, LFMADS5 into D-class, and LFMADS6-7 into E-class, respectively. LFMADS-GFP fusion proteins appeared to localize in the nucleus, supporting their roles as transcription factors (TFs). Overexpression of the LFMADS2, LFMADS4, and LFMADS6 genes in Arabidopsis resulted in early flowering and floral defect, however, only early flowering in transgenic tobacco was observed. Highly expressed floral integrator genes, including AtFT, AtLFY, and AtFUL in transgenic Arabidopsis and NtFUL and NtSOC1 in transgenic tobacco, resulted in early flowering phenotype through qRT-PCR analysis. Yeast two-hybrid analysis suggested that LFMADSs may form higher order complexes with the B-, C-, D, and/or E-class proteins to determine the floral organ identity. Furthermore, E-class LFMADS proteins may function as a glue to mediate and strengthen the protein-protein interactions. Therefore, our de novo datasets would provide information for investigating other differentially expressed candidate transcripts. In addition, functional conservation of LFMADSs appears to be vital in floral transition and floral organ identity.

Identification of Heat Shock Transcription Factor Genes Involved in Thermotolerance of Octoploid Cultivated Strawberry.

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a, which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress.

Chloroplast Protein Tic55 Involved in Dark-Induced Senescence through AtbHLH/AtWRKY-ANAC003 Controlling Pathway of Arabidopsis thaliana.

The chloroplast comprises the outer and inner membranes that are composed of the translocon protein complexes Toc and Tic (translocon at the outer/inner envelope membrane of chloroplasts), respectively. Tic55, a chloroplast Tic protein member, was shown to be not vital for functional protein import in Arabidopsis from previous studies. Instead, Tic55 was revealed to be a dark-induced senescence-related protein in our earlier study. To explore whether Tic55 elicits other biological functions, a tic55-II knockout mutant (SALK_086048) was characterized under different stress treatments. Abiotic stress conditions, such as cold, heat, and high osmotic pressure, did not cause visible effects on tic55-II mutant plant, when compared to the wild type (WT). In contrast, senescence was induced in the individually darkened leaves (IDLs), resulting in the differential expression of the senescence-related genes PEROXISOME DEFECTIVE 1 (PED1), BLUE COPPER-BINDING PROTEIN (BCB), SENESCENCE 1 (SEN1), and RUBISCO SMALL SUBUNIT GENE 2B (RBCS2B). The absence of Tic55 in tic55-II knockout mutant inhibited expression of the senescence-related genes PED1, BCB, and SEN1 at different stages of dark adaptation, while causing stimulation of RBCS2B gene expression at an early stage of dark response. Finally, yeast one-hybrid assays located the ANAC003 promoter region with cis-acting elements are responsible for binding to the different AtbHLH proteins, thereby causing the transactivation of an HIS3 reporter gene. ANAC003 was shown previously as a senescence-related protein and its activation would lead to expression of senescence-associated genes (SAGs), resulting in plant senescence. Thus, we propose a hypothetical model in which three signaling pathways may be involved in controlling the expression of ANAC003, followed by expression of SAGs that in turn leads to leaf senescence in Arabidopsis by this study and previous data.

Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1.

Chemokine CXCL8 is crucial for regulation of inflammatory and immune responses via activating its cognate receptor CXCR1. In this study, molecular docking and binding free energy calculations were combined to predict the initial binding event of CXCL8 to CXCR1 for peptide drug design. The simulations reveal that in the initial binding, the N-loop of CXCL8 interacts with the N-terminus of CXCR1, which is dominated by electrostatic interactions. The derived peptides from the binding region of CXCL8 are synthesized for further confirmation. Surface plasmon resonance analyses indicate that the CXCL8 derived peptide with 14 residues is able to bind to the receptor CXCR1 derived peptide with equilibrium KD of 252 µM while the peptide encompassing a CXCL8 K15A mutation hardly binds to CXCR1 derived peptide (KD = 1553 µM). The cell experiments show that the designed peptide inhibits CXCL8-induced and LPS-activated monocytes adhesion and transmigration. However, when the peptides were mutated on two lysine residues (K15 and K20), the inhibition effects were greatly reduced indicating these two amino acids are key residues for the initial binding of CXCL8 to CXCR1. This study demonstrates that in silico prediction based functional peptide design can be effective for developing anti-inflammation drugs.

Unique features of Erwinia chrysanthemi (Dickeya dadantii) RA3B genes involved in the blue indigoidine production.

Erwinia chrysanthemi (Ech) RA3B produces a large amount of blue indigoidine. Using Tn5-induced mutagenesis, three indigoidine-deficient mutants were generated. Followed by library screening, a 5.8kb fragment complemented mutants for indigoidine synthesis was cloned. This fragment contains four complete open-reading frames (ORFs), pecS, pecM, idgA, and idgB, and two partial ORFs, argG, and idgC. These genes are nearly identical to those in strain Ech3937. Primer extension assays demonstrated a clear transcriptional start site prior to idgA, while no promoter preceding idgB and idgC was detected, suggesting that idgA, idgB, and idgC are organized as one transcription unit. In contrast, indAB is separated from indC in Ech3937. Interestingly, an ERIC sequence was present between idgB and idgC in place of the promoter region of the homolog indC, which may contribute to the loss of promoter activity in RA3B. Futhermore, idgB mutant displayed much lighter blue color, while indB mutant appeared white on media. Overexpression of pecS in RA3B resulted in significantly reduced indigoidine production and idgC transcript. Moreover, gel shift and luxAB reporter assays revealed that PecS specifically binds to the sequence preceding idgA and inhibits gene expression, which is consistent with the results observed in Ech3937.

A system for the enhancement of adenovirus mediated gene transfer to uro-epithelium.

PURPOSE: Recombinant adenovirus has been used widely as an in vivo gene transfer vector, although its transfection efficiency in bladder tissue is limited. Several studies have indicated that the bladder surface glycosaminoglycan (GAG) layer functions as a nonspecific anti-adherence factor and possibly as a first line anti-infection defense mechanism. We determined whether recombinant adenovirus mediated gene transfer could be enhanced in intact bladders by HCl pretreatment and by alterations in the GAG layer. MATERIALS AND METHODS: In vitro viral transfection efficiencies with and without the GAG analog pentosan polysulfate (Sigma Chemical Co., St. Louis, Missouri) were determined in bladder muscle and urothelial cells. Immunocytochemical studies and Western blot analysis were performed to determine whether urothelial cells possessed the Coxsackievirus and adenovirus receptor. Rat bladders were intravesically pretreated with HCl at various concentrations and for various periods. After 60 mM. HCl pretreatment for 10 minutes 2 x 109 pfu of recombinant adenovirus carrying the Escherichia coli LacZ gene were intravesically instilled into the bladders. RESULTS: Adenoviral infection of urothelial cells was significantly reduced in the presence of pentosan polysulfate in vitro. Coxsackievirus and adenovirus receptor expression was detected in urothelial cells in vivo and in vitro. Bladders pretreated with HCl resulted in an alteration of the bladder GAG layers. After intravesical gene instillation reporter gene analyses using X-5-bromo-4-chloro-3-inodolyl beta-D-galactopyranoside (Sigma Chemical Co.) showed approximately 80% urothelial cell transfection efficiency in bladders pretreated with HCl. However, less than 10% of the urothelial cells expressed the transfected gene in control HCl untreated bladders. CONCLUSIONS: Primary urothelial cells and bladder carcinoma cells can be efficiently transfected using an adenoviral vector with similar infectivity. In vitro viral infection shows that the efficiency of adenoviral transfection is significantly reduced in the presence of pentosan polysulfate, a GAG analog. Adenoviral mediated gene transfer to bladder urothelium is enhanced by HCl pretreatment.