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Background Leaf senescence comprises numerous cooperative events, integrates environmental signals with age-dependent developmental cues, and coordinates the multifaceted deterioration and source-to-sink allocation of nutrients. In crops, leaf senescence has long been regarded as an essential developmental stage for productivity and quality, whereas functional characterization of candidate genes involved in the regulation of leaf senescence has, thus far, been limited in wheat. Results In this study, we analyzed the expression profiles of 97 WRKY transcription factors (TFs) throughout the progression of leaf senescence in wheat and subsequently isolated a potential regulator of leaf senescence, TaWRKY42-B, for further functional investigation. By phenotypic and physiological analyses in TaWRKY42-B-overexpressing Arabidopsis plants and TaWRKY42-B-silenced wheat plants, we confirmed the positive role of TaWRKY42-B in the initiation of developmental and dark-induced leaf senescence. Furthermore, our results revealed that TaWRKY42-B promotes leaf senescence mainly by interacting with a JA biosynthesis gene, AtLOX3, and its ortholog, TaLOX3, which consequently contributes to the accumulation of JA content. In the present study, we also demonstrated that TaWRKY42-B was functionally conserved with AtWRKY53 in the initiation of age-dependent leaf senescence. Conclusion Our results revealed a novel positive regulator of leaf senescence, TaWRKY42-B, which mediates JA-related leaf senescence via activation of JA biosynthesis and has the potential to be a target gene for molecular breeding in wheat.

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

Whether ambient temperature influences immune responses leading to uveitis is unknown. We thus tested whether ambient temperature affects the symptoms of experimental autoimmune uveitis (EAU) in mice and investigated possible mechanisms. C57BL/6 mice were kept at a normal (22 degrees C) or high temperature (30 degrees C) housing conditions for 2 weeks and were then immunized with human interphotoreceptor retinoid-binding protein (IRBP651-670) peptide to induce EAU. Histological changes were monitored to evaluate the severity of uveitis. Frequency of Th1 cells and Th17 cells was measured by flow cytometry (FCM). The expression of IFN-gamma and IL-17A mRNA was measured by real-time qPCR. The generation of neutrophil extracellular traps (NETs) was quantified by enzyme-linked immunosorbent assay (ELISA). Differential metabolites in the plasma of the mice kept in the aforementioned two ambient temperatures were measured via ultra-high-performance liquid chromatography triple quadrupole mass spectrometry quadrupole time of flight mass spectrometry (UHPLC-QQQ/MS). The differential metabolites identified were used to evaluate their effects on differentiation of Th1 and Th17 cells and generation of NETs in vitro. The results showed that EAU mice kept at high temperature experienced a more severe histopathological manifestation of uveitis than mice kept at a normal temperature. A significantly increased frequency of Th1 and Th17 cells in association with an upregulated expression of IFN-gamma and IL-17A mRNA was observed in the splenic lymphocytes and retinas of EAU mice in high temperature. The expression of NETs as evidenced by myeloperoxidase (MPO) and neutrophil elastase (NE), was significantly elevated in serum and supernatants of neutrophils from EAU mice kept at high temperature compared to the normal temperature group. The metabolites in the plasma from EAU mice, fumaric acid and succinic acid, were markedly increased in the high temperature group and could induce the generation of NETs via the NADPH oxidase-dependent pathway, but did not influence the frequency of Th1 and Th17 cells. Our findings suggest that an increased ambient temperature is a risk factor for the development of uveitis. This is associated with the induction of Th1 and Th17 cells as well as the generation of NETs which could be mediated by the NADPH oxidase-dependent pathway.

Background: Vogt-Koyanagi-Harada (VKH) disease is a multisystemic autoimmune disorder characterized by granulomatous panuveitis. Gut microbiome has been considered to play a role in the pathogenesis of this disease but whether the alternation of gut microbiome was involved is unclear. This study was set up to identify abnormalities of gut microbiome composition in VKH disease. Results: Depleted butyrate-producing bacteria, lactate-producing bacteria and methanogens as well as enriched Gram-negative bacteria were identified in the active VKH patients, as well as in VKH patients of Mix enterotype and Bacteroides enterotype. Changes of gut microbiome in the VKH patients were partially restored after an immunosuppressive treatment. The disease susceptibility genotype HLA-DRA was associated with Bacteroides sp.2.1.33B, Paraprevotella clara, Alistipes finegoldii and Eubacterium eligens. A microbial marker profile including 40 disease-associated species was established to differentiate patients from controls. Another microbial marker profile including 37 species was found to be associated with the response to treatment. An animal experiment showed that transfer of gut microbiome from VKH patients could significantly exacerbate disease activity clinically and pathologically in the recipient mice. Conclusion: Our results revealed a distinct gut microbiome signature in VKH patients and showed an exacerbating effect of this gut microbiome on experimental autoimmune uveitis (EAU). We also developed two microbial marker profiles in differentiating VKH patients from healthy controls as well as predicting the effectiveness of treatment.

Leaf senescence is a complex and precise regulatory process that is correlated with numerous internal and environmental factors. Leaf senescence is tightly related to the redistribution of nutrients, which significantly affects productivity and quality, especially in crops. Evidence shows that the mediation of transcriptional regulation by WRKY transcription factors is vital for the fine-tuning of leaf senescence. However, the underlying mechanisms of the involvement of WRKY in leaf senescence are still unclear in wheat. Using RNA sequencing data, we isolated a novel WRKY transcription factor, TaWRKY40-D, which localizes in the nucleus and is basically induced by the progression of leaf senescence. TaWRKY40-D is a promoter of natural and dark-induced leaf senescence in transgenic Arabidopsis thaliana and wheat. We also demonstrated a positive response of TaWRKY40-D in wheat upon jasmonic acid (JA) and abscisic acid (ABA) treatment. Consistent with this, the detached leaves of TaWRKY40-D VIGS (virus-induced gene silencing) wheat plants showed a stay-green phenotype, while TaWRKY40-D overexpressing Arabidopsis plants showed premature leaf senescence after JA and ABA treatment. Moreover, our results revealed that TaWRKY40-D positively regulates leaf senescence, possibly by altering the biosynthesis and signalling of JA and ABA pathway genes. Together, our results suggest a new regulator of JA- and ABA-related leaf senescence, as well as a new candidate gene that can be used for molecular breeding in wheat.

Background: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism?Methods: EAU was induced in B1 OR.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis.Results: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones.Conclusions: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

AimTo elucidate the role of microRNA-20a-5p (miR-20a-5p) in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.MethodsQuantitative real-time PCR was used to quantify miR-20a-5p expression in CD4+ T cells from patients with active VKH and normal controls. The promoter methylation status of miR-20a-5p was detected by bisulfite sequencing PCR. Targets were evaluated by a luciferase reporter assay. The functional effects of miR-20a-5p on CD4+ T cells from patients with active VKH were assessed by upregulation or downregulation of its expression using liposomes.ResultsThe miR-20a-5p level was significantly decreased in CD4+ T cells from patients with active VKH as compared with normal controls. The two genes, oncostatin M (OSM) and C-C motif chemokine ligand 1 (CCL1), were identified as targets of miR-20a-5p. The upregulation of miR-20a-5p significantly suppressed interleukin 17 (IL-17) production in CD4+ T cells from patients with active VKH, whereas downregulation of miR-20a-5p exhibited an inverse effect. In addition, overexpression of OSM and CCL1 could rescue the effect of the upregulation of miR-20a-5p. Moreover, the level of miR-20a-5p was reduced in response to hypermethylation of the promoter. Further study showed that miR-20a-5p suppressed the activity of the phosphoinositide 3-kinase-AKT pathway.ConclusionsOur findings indicate that downregulation of miR-20a-5p is caused by promoter hypermethylation. MiR-20a-5p could also suppress the production of IL-17 by targeting OSM and CCL1 production in CD4+ T cells in patients with active VKH.

Leaf senescence is a positive, highly regulated, complex process, and transcription factors play important roles in the regulation of this process. We identified and characterized 116 WRKYs from the wheat genome database. Thirteen TaWRKYs were confirmed as senescence-associated genes. We focused on TaWRKY7, which is up-regulated in the natural leaf senescence process. TaWRKY7 is expressed in different tissues of wheat and is localized in the nucleus. It shows transcriptional activation activity in yeast cells. The ectopic over-expression of TaWRKY7 in Arabidopsis (Arabidopsis thaliana) significantly promoted early leaf senescence under darkness treatment and prevented leaf moisture losses. TaWRKY7 played important roles in the senescence process and was involved in abiotic stress responses. Our transcriptomic and genetic studies on WRKYs suggest that WRKY transcription factors are a type of vital regulator in leaf senescence in wheat (Triticum aestivum L.).

PURPOSE. Recent studies reported that the tumor suppressor disabled-2 (DAB2) is a negative regulator of immune function. In this study, we investigated the role of DAB2 in monocyte-derived dendritic cells (DCs) from Vogt-Koyanagi-Harada disease (VKH) patients. METHODS. The mRNA and protein levels of DAB2 were quantified by quantitative real-time PCR and Western blot. The Sequenom MassARRAY system was used to detect the promoter methylation level. An adenovirus carrying the DAB2 gene was transduced into immature DCs, isolated, and induced from active VKH patients. The surface markers of DCs, the frequency of T helper (Th) type 1 (Th1) and Th17 cells in CD4(+)T cells, which were cocultured with DCs, were tested by flow cytometry. ELISA was used to analyze the inflammatory cytokines produced by DC and CD4(+) T cell cocultures. RESULTS. The mRNA and protein expression levels of DAB2 in DCs obtained from active VICH patients were decreased, while the DAB2 promoter methylation level was marginally increased when compared with inactive VKH patients and normal controls. The expression of CD86 on DCs was significantly downregulated by DAB2 overexpression. The DC-related inflammatory factors IL-6 and TNE-alpha were also decreased. The frequency of Th1 and Th17 cells and their related cytokines were reduced significantly after coculture with DAB2 overexpressing DCs. DAB2 overexpression did not affect autophagy in DCs from VKH patients. CONCLUSIONS. These results suggest that the decreased expression of DAB2 in DCs plays a role in the pathogenesis of VKH disease. DAB2 overexpression inhibits DC function, but this is not mediated via autophagy.

Background Behcet’s disease (BD) is a recalcitrant, multisystemic inflammatory disease that can lead to irreversible blindness. Microbial agents have been considered to contribute to the pathogenesis of this disease, but the underlying mechanisms remain unclear. In this study, we investigated the association of gut microbiome composition with BD as well as its possible roles in the development of this disease. Methods Fecal and saliva samples were collected from 32 active BD patients and 74 healthy controls. DNA extracted from fecal samples was subjected to metagenomic analysis, whereas DNA extracted from saliva samples was subjected to 16S rRNA gene sequencing analysis. The results were used to compare the composition and biological function of the microbiome between patients and healthy controls. Lastly, transplantation of pooled fecal samples from active BD patients into B10RIII mice undergoing experimental autoimmune uveitis (EAU) was performed to determine the causal relationship between the gut microbiome and BD. Results Fecal samples from active BD patients were shown to be enriched in Bilophila spp., a sulfate-reducing bacteria (SRB) and several opportunistic pathogens (e.g., Parabacteroides spp. and Paraprevotella spp.) along with a lower level of butyrate-producing bacteria (BPB) Clostridium spp. and methanogens (Methanoculleus spp. Methanomethylophilus spp.). Analysis of microbial functions revealed that capsular polysaccharide transport system, oxidation-reduction process, type III, and type IV secretion systems were also increased in active BD patients. Network analysis showed that the BD-enriched SRB and opportunistic pathogens were positively correlated with each other, but they were negatively associated with the BPB and methanogens. Animal experiments revealed that fecal microbiota transplantation with feces from BD patients significantly exacerbated EAU activity and increased the production of inflammatory cytokines including IL-17 and IFN-γ. Conclusions Our findings revealed that BD is associated with considerable gut microbiome changes, which is corroborated by a mouse study of fecal microbiota transplants. A model explaining the association of the gut microbiome composition with BD pathogenesis is proposed. Electronic supplementary material The online version of this article (10.1186/s40168-018-0520-6) contains supplementary material, which is available to authorized users.

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells’ signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics.

Senescence is the final stage of leaf development. During this process, different macromolecules undergo degradation, and the resulting components are transported to developing and storage tissues of the plant. Senescence-associated genes (SAGs) play important roles in this process. Identification and characterization of SAGs are the first steps to interpret the function of these genes and to elucidate the mechanisms of leaf senescence. One of the most effective ways to identify SAGs is to screen for candidate genes using massive genome-scale transcriptomic data such as microarray, RNA-seq, digital RNA expression level data, etc. The basic functional analysis of candidate genes is to observe the phenotypes of transgenic plants, in which the candidate SAGs are overexpressed, knocked down, or knocked out. In this chapter, we outline methods for identifying and characterizing SAGs by microarray analysis in wheat. Methods of gene functional analyses by screening transgenic plants are also described. The protocols described in this chapter could also be used in other plant species, especially for Poaceae plants.

The foxtail millet NAC transcription factor NAC1, an ortholog of Arabidopsis NAP, is induced by ABA and senescence and accelerates leaf senescence by promoting ABA biosynthesis. Leaf senescence, a unique developmental stage involving macromolecule degradation and nutrient remobilization, is finely tuned and tightly controlled by different gene families. NO APICAL MERISTEM, ARABIDOPSIS ATAF1, and CUP-SHAPED COTYLEDON (NAC) transcription factors have been demonstrated to be involved in the modulation of leaf senescence in many land plant species. Foxtail millet (Setaria italica L.), an important food and fodder crop, has been studied for its strong stress tolerance and potential to be a biofuel model plant. However, the functional roles of senescence-associated NACs in foxtail millet are still unknown. In this study, we characterized a nuclear localized NAC transcription factor, SiNAC1, which is induced by senescence and concentrated in senescent leaves in foxtail millet. SiNAC1 also positively responds to abscisic acid (ABA) treatment in foxtail millet. Moreover, SiNAC1 promotes the natural and dark-induced leaf senescence by an ABA-dependent manner in Arabidopsis thaliana. NCED2 and NCED3 are elevated by SiNAC1 overexpression, which subsequently promotes ABA biosynthesis in Arabidopsis. Finally, as a homolog of AtNAP, SiNAC1 can partially rescue the delayed leaf senescence phenotype in atnap mutants. Overall, our results demonstrate that SiNAC1 functions as a positive regulator of leaf senescence and is involved in a positive feedback loop via ABA biosynthesis and leaf senescence.

Leaf senescence is induced or accelerated when leaves are detached. However, the senescence process and expression pattern of senescence-associated genes (SAGs) when leaves are detached are not clearly understood. To detect senescence-associated physiological changes and SAG expression, wheat (Triticum aestivum L.) leaves were detached and treated with light, darkness, low temperature (4 °C), jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA). The leaf phenotypes, chlorophyll content, delayed fluorescence (DF), and expression levels of two SAGs, namely, TaSAG3 and TaSAG5, were analyzed. Under these different treatments, the detached leaves turned yellow with different patterns and varying chlorophyll content. DF significantly decreased after the dark, ABA, JA and SA treatments. TaSAG3 and TaSAG5, which are expressed in natural senescent leaves, showed different expression patterns under various treatments. However, both TaSAG3 and TaSAG5 were upregulated after leaf detachment. Our results revealed senescence-associated physiological changes and molecular differences in leaves, which induced leaf senescence during different stress treatments.

TaGSK1, a salt-inducible gene, has a very high identity with mammalian GSK3 which is a highly conserved serine/threonine protein kinase that regulates glycogen synthesis. Overexpression of TaGSK1 reduces cellular osmotic turgor and cellular Na+ content and enhances salt tolerance of transgenic Arabidopsis plants. Biochemical analysis of transgenic lines of Arabidopsis has revealed that the binding property of TaGSK1 protein to CaM was Ca2+ dependent, and the TaGSK1 protein has both Ca2+- and CaM-independent self-phosphorylation and substrate phosphorylation kinase activities. Genomic DNA sequence analysis of wheat has revealed that TaGSK1 gene contains a 703-bp-long intron in the 5′ non-coding region that significantly enhances gene expression and is capable of activating GUS gene transcription. The promoter of TaGSK1 is confirmed to be salt-inducible. The mechanism for salt tolerance mechanism conferred by TaGSK1 was discussed.

Summary T helper type 2 (Th2) and regulatory T cells (Treg) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce Treg cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and Treg cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/Treg cells.

Purpose To investigate gut microbiota composition and fecal metabolic phenotype in patients with acute anterior uveitis. Methods Fecal DNA was extracted from 78 fecal samples (38 acute anterior uveitis (AAU) patients and 40 family members of patients or sex- and age-matched healthy controls) and then sequenced by high-throughput 16S rDNA analysis. Gas chromatographic mass spectrometry (GC-MS) based metabolomics was performed on 60 fecal samples (30 AAU patients and 30 healthy controls). Results A significant difference was observed in beta diversity between AAU patients and healthy controls. Eight genera including Roseburia were reduced in AAU patients, and Veillonella was increased in AAU patients as compared with healthy controls. Significance was however lost after false discovery rate (FDR) correction. The expression of seven fecal metabolites including 6-deoxy-D-glucose 1, linoleic acid, N-Acetyl-beta-D-mannosamine 3, shikimic acid, azelaic acid, isomaltose 1 and palmitoleic acid was increased in AAU patients. Linoleic acid showed a significant correlation with Roseburia and Veillonella according to Spearman correlation analysis. Conclusions Our results did not reveal a difference in gut microbiota composition, but did show that the fecal metabolic phenotype in AAU patients was significantly different from healthy controls.

High soil salinity is a major abiotic stress in plant agriculture worldwide. Here, we report the characterization of a novel aquaporin gene TaNIP (Triticum asetivum L. nodulin 26-like intrinsic protein), which was involved in salt tolerance pathways in plants. TaNIP was identified and cloned through the gene chip expression analysis of a salt-tolerant wheat mutant RH8706-49 under salt stress. Quantitative reverse transcription-PCR (Q-RT-PCR) was used to detect TaNIP expression under salt, drought, cold and ABA treatment. The overexpression of TaNIP in transgenic Arabidopsis produced higher salt tolerance than wild-type plants. Localization analysis showed that TaNIP proteins tagged with green fluorescent protein (GFP) were localized to the cell plasma membrane. Under salt stress treatment, TaNIP-overexpressing Arabidopsis accumulated higher K(+), Ca(2+) and proline contents and lower Na(+) level than the wild-type plants. The overexpression of TaNIP in transgenic Arabidopsis also up-regulated the expression of a number of stress-associated genes. Our results suggest that TaNIP plays an important role in salt tolerance in Arabidopsis and can also enhance plants' tolerance to other abiotic stresses.

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development, and it can be regulated by multiple environmental cues and endogenous signals. The mitogen-activated protein kinase (MAPK) cascades play diverse roles in intracellular and extracellular signaling in plants. Roles of the MAPK signaling module in leaf senescence are unknown. Here, a MAPK cascade involving MKK9-MPK6 is shown to play an important role in regulating leaf senescence in Arabidopsis (Arabidopsis thaliana). Both MKK9 and MPK6 possess kinase activities, with MPK6 an immediate target of MKK9, as revealed by in vitro, in vivo, and in planta assays. The constitutive and inducible overexpression of MKK9 causes premature senescence in leaves and in whole Arabidopsis plants. The premature senescence phenotype is suppressed when MKK9 is overexpressed in the mpk6 null background. When either MKK9 or MPK6 is knocked out, leaf senescence is delayed.

In comparison to herbaceous plants, studies of cold responsive genes and cold signaling in woody perennials are limited. Since most woody plants have evolved freezing tolerance (FT) in winter, together with similar lignified structures and winter adaptive mechanisms, it is more likely to find cold resistant genes in woody plants growing in temperate and arctic regions. In this study, Ammopiptanthus mongolicus, an evergreen, broadleaf and leguminous shrub was selected as a model to study cold tolerance in woody plants. Thirteen cold up-regulated cDNAs (including eight full-length cDNAs and five partial cDNAs) were cloned from A. mongolicus. One of these genes, AmEBP1, confers enhanced cold tolerance to E. coli and obvious increased freezing survival to Arabidopsis. In transgenic Arabidopsis expressing AmEBP1, transcript levels of some downstream genes in cold signaling exhibit increased accumulation upon cold treatment. Together with structural information, sub-cellular location, and promoter analysis data, it is suggested that AmEBP1 enhances plants cold tolerance by accelerating ribosome biogenesis and the concomitant translation of cold induced transcription factors and downstream protective proteins under cold stress.

A putative isopentenyltransferase (IPT) encoding gene was identified from the soybean expressed sequence tag (EST) database, and the full-length gene was cloned with RACE. The gene was most similar to AtIPT5, an isopentenyltransferase gene in Arabidopsis, with respect to expression profile and sequence alignment. Southern hybridization showed that there were two copies (isoforms) of GmIPT in the soybean genome. Transgenic study with tobacco in the present study indicated that the protein encoded by the GmIPT1 gene could produce active cytokinins (CKs) in plants. RT-PCR revealed that the GmIPT1 gene may be expressed constitutively in several tissues. Promoter–GUS fusion revealed highly specific expression sites of GmIPT1 in roots, shoots and leaves, although these were not detected by Northern blot analysis, due to very low expression status. The isolated upstream regulatory sequence of GmIPT1 shared common transcription factor binding motifs with the corresponding regulatory regions of AtIPTs and OsIPTs. GmIPT1 expression was up-regulated by other phytohormones and chemicals, as indicated by the results of quantitative PCR and GUS activity examination in transgenic Arabidopsis. This is consistent with finding putative phytohormone and stress-responsive motifs in the promoter region. Involvement of putative signals and/or other factors in regulating GmIPT1 expression, and related control of CK production in higher plants are discussed.

A rice gene, OsAPT2, which encodes a putative adenine phosphoribosyl transferase (APRT), was cloned and characterized. Analysis of the cDNA and genomic sequences revealed seven exons and six introns in the OsAPT2. The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously isolated APRTs. RT-PCR analysis indicated that the OsAPT2 transcript in the young panicles of 'Annong S-1' is down-regulated at 29 degrees C, the critical temperature for induction of 'Annong S-1' fertility conversion. Since the panicle is likely the thermo-sensitive organ at the early stages of pollen fertility alternation, the observed heat-induced change in the OsAPT2 expression pattern in young panicles may mediate, at least in part, thermo-sensitive genic male sterility (TGMS) in 'Annong S-1'. An antisense strategy was used to suppress the expression of the OsAPT2 homolog in Arabidopsis, and the obtained homozygous transgenic plants contained lower AMP content, displayed lower pollen germination rates and exhibited some abnormalities in leaf phenotypes and flowering timing. These data suggest that OsAPT2 is likely to be involved in TGMS in the rice line 'Annong S-1'.

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

Adenine phosphoribosyl transferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. A novel form of APRT gene from wheat (Triticum aestivum L.) was cloned through a combination of bioinformatic, RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The total length of the cDNA sequence is 1097 nucleotides, in which an open reading frame (ORF) was found that encodes 221 amino acid residues. Alignment of the deduced amino acid sequence and that of the other plant APRT genes indicates that the novel cDNA in full-length is the form 2 of wheat APRT gene, which was named as TaAPT2. Twelve Single Nucleotide Polymorphism (SNP) sites were identified in the TaAPT2 gene from BNY-S, one wheat temperature-sensitive genic male sterile (TGMS) mutant line derived from the wild type of BNY-F, among which a single base substitution in the purine-binding domain resulted in the Asn to Thr transition. The protein secondary structure prediction revealed that this SNP mutation subsequently resulted in many changes in the quantity and the positions of the helix, extended strand and the loop, which perhaps affect the combining efficiency of the APRT. Northern analysis indicated that the abundance of TaAPT2 gene transcript in the young spikelets reduced obviously in BNY-S under low temperature stress (lower than 10 °C) at the early stage of pollen fertility alternation, however, the transcript of TaAPT2 gene in BNY-F was not affected, indicating that wheat TaAPT2 gene may be related to the fertility alternation and abortion of pollen development.

The genusPopulus L. (Salicaceae) can be divided into 5 sections with distribution throughout the world. Accurate identification ofPopulus clones and species is essential for effective selection, breeding, and management of genetic resources. In this study, amplified fragment length polymorphism (AFLP) analysis, which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct analyses of genetic diversity and variety identification of 44 species, clones, and cultivars ofPopulus that represent a wide range of breeding and commercially available germplasms. Cluster analysis of the 44 samples was carried out, and a dendrogram of genetic relatedness was developed on the basis of the AFLP data. DNA fingerprints of the 44 samples were developed from 12 selected bands amplified with 2 primer combinations (M-CAG/E-TA and M-CAG/E-TC). Each sample has its unique fingerprint pattern and can be distinguished from the others. Furthermore, 1 specific AFLP band of the cultivarPopulus canadensis cl. Guariento coming from fragments amplified by primer combination M-CTC/E-AG was successfully converted into a sequence-characterized amplified region (SCAR) marker. The results indicate that AFLP analysis should be considered as the preferred technique for the study of polymorphism inPopulus. This research is the first report concerning the use of AFLP analysis in genetic diversity and germplasm identification among all sections ofPopulus.

Polygalacturonase-inhibiting Proteins (PGIPs) are plant proteins that counteract fungal Polygalacturonases (PGs), which are important virulence factors. As defense proteins, PGIP can efficiently limit fungal invasion. In this study, a full-length cDNA of PPGIP1 gene (peach PGIP gene copy no.1) has been cloned from peach [prunus persica (L.) Batch] variety ‘Okubo’, which is resistant to fungal diseases such as peach scab. Sequence analysis revealed that the cDNA of PPGIP1 is 1380 bp in length and that its containing open reading frame (ORF) encodes for a polypeptide of 330 amino acids. The PPGIP1 gene from genomic DNA shows a single 147 bp intron that is efficiently spliced out of the PPGIP1 pre-mRNA transcript. The PPGIP1 has a high degree of identify with previously isolated PGIP genes and the encoded polypeptide shows all the characteristic features of PGIP peptides. Analysis of genomic organization and expression indicate that the PPGIP1 gene is one of a low copy number gene family with three copies in peach genome and its expression can be induced by exogenous salicylic acid (SA).

A thermo-sensitive genic male-sterile (TGMS) wheat line ( Triticum aestivum L.) BNY-S was obtained from the spontaneous mutant of BNY-F. Its fertility was decided by the temperature during the differentiation stage of the spikelets. BNY-S was completely sterile when the temperature was lower than 10 degrees C during the differentiation stage of the spikelets, but fertile when the temperature was higher than 10 degrees C. Genetic analysis indicated that the sterility of BNY-S was controlled by a single recessive gene, which was named as wtms1. An F(2) population, consisting of 3,000 individuals from the cross between BNY-S and Lankao 52-24, was used for genetic analysis and statistical analysis of the TGMS and, out of them, 158 sterile and 93 fertile extremes were present for molecular tagging and mapping of the wtms1 gene. SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) techniques combined with BSA (bulked segregant analysis) were used to screen markers linked to the target gene. As a result, wtms1 was preliminarily mapped on chromosome 2B according to SSR analysis. In AFLP analysis, 14 polymorphic AFLP loci were identified with a linkage relation to the wtms1 gene. Then linkage analysis using the F(2) population showed that three of them, E: AAG/M: CTA(163), E: AGG/M: CTC(220) and E: ACA/M: CTA(160), were linked to the wtms1 gene relatively close to a genetic distance of 6.9 cM, 6.9 cM and 13.9 cM, respectively. Finally, the wtms1 gene was mapped between the SSR marker Xgwm 374 and the AFLP marker E: AAG/M: CTA(163) with the distance of 4.8 cM and 6.9 cM, respectively. A partial linkage map was constructed according the SSR and AFLP data.

Leaf senescence is a complex developmental phase that involves both degenerative and nutrient recycling processes. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. The onset and progression of leaf senescence are controlled by an array of environmental cues (such as drought, darkness, extreme temperatures, and pathogen attack) and endogenous factors (including age, ethylene, jasmonic acid, salicylic acid, abscisic acid, and cytokinin). This review discusses the major breakthroughs in signal transduction during the onset of leaf senescence, in dark- and drought-mediated leaf senescence, and in various hormones regulating leaf senescence achieved in the past several years. Various signals show different mechanisms of controlling leaf senescence, and cross-talks between different signaling pathways make it more complex. Key senescence regulatory networks still need to be elucidated, including cross-talks and the interaction mechanisms of various environmental signals and internal factors.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.