Isolation and functional analysis of SrMYB1,a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.

Insights into cryptic speciation of quillworts in China

Cryptic species are commonly misidentified because of high morphological similarities to other species.One group of plants that may harbor large numbers of cryptic species is the quillworts(Isoetes spp.),an ancient aquatic plant lineage.Although over 350 species of Isoetes have been reported globally,only ten species have been recorded in China.The aim of this study is to better understand Isoetes species diversity in China.For this purpose,we systematically explored the phylogeny and evolution of Isoetes using complete chloroplast genome(plastome)data,spore morphology,chromosome number,genetic structure,and haplotypes of almost all Chinese Isoetes populations.We identified three ploidy levels of Isoetes in Chinaddiploid(2n=22),tetraploid(2n=44),and hexaploid(2n=66).We also found four megaspore and microspore ornamentation types in diploids,six in tetraploids,and three in hexaploids.Phylogenetic analyses confirmed that I.hypsophila as the ancestral group of the genus and revealed that Isoetes diploids,tetraploids,and hexaploids do not form monophyletic clades.Most individual species possess a single genetic structure;however,several samples have conflicting positions on the phylogenetic tree based on SNPs and the tree based on plastome data.All 36 samples shared 22 haplotypes.Divergence time analysis showed that I.hypsophila diverged in the early Eocene(~48.05 Ma),and most other Isoetes species diverged 3-20 Ma.Additionally,different species of Isoetes were found to inhabit different water systems and environments along the Yangtze River.These findings provide new insights into the relationships among Isoetes species in China,where highly similar morphologic populations may harbor many cryptic species.

Phosphorylation regulation of nitrogen,phosphorus,and potassium uptake systems in plants

The uptake of ammonium,nitrate,phosphorus,and potassium ions by roots is mediated by specific ion transporter or channel proteins,and protein phosphorylation regulation events occurring on these proteins and their regulators determine their ultimate activity.Elucidating the mechanism by which protein phosphorylation modification regulates nutrient uptake will advance plant breeding for high nutrientuse efficiency.In this review,it is concluded that the root nutrient absorption system is composed of several,but not all,members of a specific ion transporter or channel family.Under nutrient-starvation conditions,protein phosphorylation-based regulation of these proteins and associated transcription factors increases ion transporter-or channel-mediated nutrient uptake capacity via direct function activity enhancement,allowing more protein trafficking to the plasma membrane,by strengthening the interaction of transporters and channels with partner proteins,by increasing their protein stability,and by transcriptional activation.Under excessive nutrient conditions,protein phosphorylation-based regulation suppresses nutrient uptake by reversing these processes.Strengthening phosphorylation regulation items that increase nutrient absorption and weakening phosphorylation modification items that are not conducive to nutrient absorption show potential as strategies for increasing nutrient use efficiency.

The pentatricopeptide repeat protein EMP601 functions in maize seed development by affecting RNA editing of mitochondrial transcript ccmC

Although several pentatricopeptide repeat(PPR) proteins are involved in post-transcriptional processing of mitochondrial RNA, it is unclear which specific protein is involved in the RNA editing of ccmC in maize(Zea mays). Here we report the identification of the maize empty pericarp 601(emp601) mutant and the map-based cloning of the Emp601 gene, which encodes an E2-type PPR protein that is targeted to mitochondria. A single-nucleotide deletion in the emp601 mutant caused a frameshift and introduced a premature stop codon into the predicted EMP601. This mutation was associated with reduced accumulation of mitochondrial complex Ⅲ as well as with inhibition of growth and differentiation of basal endosperm transfer layer cells, leading to final degeneration of the embryo and endosperm. We determine that loss of EMP601 function prevents the C-to-U RNA editing of the mitochondrial transcript ccmC at position 358.EMP601 binds to the ccmC transcript and directly interacts with Multiple organellar RNA editing factor 8and may be a component of the plant mitochondrial editosome. We conclude that EMP601 functions in RNA editing of mitochondrial ccmC transcripts and influences mitochondrial function and seed development.

Fire blight disease, a fast-approaching threat to apple and pear production in China

Fire blight, caused by Erwinia amylovora, is a devastating disease of apples and pears, causing enormous economic losses around the world. The disease is indigenous to North America and has spread to more than 50 countries since its discovery in 1870 s. Recent reports of the disease in China's neighboring countries, including South Korea, Kyrgyzstan, and Kazakhstan, pose great threat to the world's leading producer of apples and pears. This mini-review intends to provide an update on the disease, pathogen biology, epidemiology, and control. It will also provide some perspectives and suggestions for the apple and pear industry and growers in China, which will face the imminent threat of this devastating disease.

A new polyacetylene glycoside from the rhizomes of Atractylodes lancea

A new polyacetylene glycoside,（1,3Z,11E）-tridecatriene-7,9-diyne-5-hydroxyl-6-O-β-D-glucopyranoside,was isolated from the rhizomes of Atractylodes lancea（Thunb.） DC.Its structure was elucidated on the basis of spectral analysis.

Genetic diversity in centipedegrass[Eremochloa ophiuroides(Munro)Hack.]

Genetic diversity is the heritable variation within and among populations,and in the context of this paper describes the heritable variation among the germplasm resources of centipedegrass.Centipedegrass is an important warmseason perennial C4 grass belonging to the Poaceae family in the subfamily Panicoideae and genus Eremochloa.It is the only species cultivated for turf among the eight species in Eremochloa.The center of origin for this species is southern to central China.Although centipedegrass is an excellent lawn grass and is most widely used in the southeastern United States,China has the largest reserve of centipedegrass germplasm in the world.Presently,the gene bank in China holds~200 centipedegrass accessions collected from geographical regions that are diverse in terms of climate and elevation.This collection appears to have broad variability with regard to morphological and physiological characteristics.To efficiently develop new centipedegrass varieties and improve cultivated species by fully utilizing this variability,multiple approaches have been implemented in recent years to detect the extent of variation and to unravel the patterns of genetic diversity among centipedegrass collections.In this review,we briefly summarize research progress in investigating the diversity of centipedegrass using morphological,physiological,cytological,and molecular biological approaches,and present the current status of genomic studies in centipedegrass.Perspectives on future research on genetics and genomics and modern breeding of centipedegrass are also discussed.

MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica

The Alternaria alternata apple pathotype adversely affects apple(Malus domestica Borkh.)cultivation.However,the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood.We have previously reported that MdWRKY75 expression is upregulated by A.alternata infection in‘Sushuai’apples.In this study,we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A.alternata infection,whereas silencing this gene enhanced susceptibility to A.alternata infection.Furthermore,we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A.alternata infection.Moreover,the thickening of the cell wall enhanced the mechanical defense capabilities of apple.In addition,we found that jasmonic acid remarkably induced MdWRKY75e expression,and its levels in transgenic apple lines were elevated.These results indicate that MdWRKY75e confers resistance to the A.alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants.In conclusion,our fi ndings provide insights into the importance of MdWRKY75e for resistance to A.alternata infection in apples.

The basic helix-loop-helix transcription factor TabHLH1 increases chlorogenic acid and luteolin biosynthesis in Taraxacum antungense Kitag

Polyphenols are the main active components of the anti-inflammatory compounds in dandelion,and chlorogenic acid(CGA)is one of the primary polyphenols.However,the molecular mechanism underlying the transcriptional regulation of CGA biosynthesis remains unclear.Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase(HQT2)is the last rate-limiting enzyme in chlorogenic acid biosynthesis in Taraxacum antungense.Therefore,using the TaHQT2 gene promoter as a probe,a yeast one-hybrid library was performed,and a basic helix-loop-helix(bHLH)transcription factor,TabHLH1,was identified that shared substantial homology with Gynura bicolor DC bHLH1.The TabHLH1 transcript was highly induced by salt stress,and the TabHLH1 protein was localized in the nucleus.CGA and luteolin concentrations in TabHLH1-overexpression transgenic lines were significantly higher than those in the wild type,while CGA and luteolin concentrations in TabHLH1-RNA interference(RNAi)transgenic lines were significantly lower.Quantitative real-time polymerase chain reaction demonstrated that overexpression and RNAi of TabHLH1 in T.antungense significantly affected CGA and luteolin concentrations by upregulating or downregulating CGA and luteolin biosynthesis pathway genes,especially TaHQT2,4-coumarate-CoA ligase(Ta4CL),chalcone isomerase(TaCHI),and flavonoid-3′-hydroxylase(TaF3′H).Dual-luciferase,yeast one-hybrid,and electrophoretic mobility shift assays indicated that TabHLH1 directly bound to the bHLH-binding motifs of proTaHQT2 and proTa4CL.This study suggests that TabHLH1 participates in the regulatory network of CGA and luteolin biosynthesis in T.antungense and might be useful for metabolic engineering to promote plant polyphenol biosynthesis.

Bleeding canker of pears caused by Dickeya fangzhongdai:Symptoms,etiology and biology

Bleeding canker,a devastating disease of pear trees(Pyrus pyrifolia L.),was first reported in the 1970 s in Jiangsu,China and more recently in other provinces in China.Trees infected with bleeding canker pathogen,Dickeya fangzhongdai,develop cankers on the trunks and branches,and a rust-colored mixture of bacterial ooze and tree sap could be seen all over the trunks and branches.In this study,we provided detail descriptions of the symptoms and epidemiology of bleeding canker disease.Based on pathogenic and phenotypic characterizations,we identified the causal agent of bleeding canker of pear as D.fangzhongdai.Dickeya fangzhongdai strains isolated from pear were also pathogenic on Solanum tuberosum,Brassica pekinensis,Lycopersicon esculentum,and Phalaenopsis aphrodite based on artificial inoculation,and the pathogen were more virulent on potato than that of D.solani strain.This study provides new information about this disease and bleeding canker disease of pear.

Neolignan glycoside from Angelica dahurica

A new neolignan glycoside, 4-O-β-d-glucopyranosyl-9-O-β-d-glucopyranosyl-(7R,8S)-dehydrodiconiferyl alcohol was isolated from the fresh roots of Angelica dahurica. The structure of the new compound was elucidated on the basis of spectral analysis.

Genomic characteristics of Dickeya fangzhongdai isolates from pear and the function of type Ⅳ pili in the chromosome

Dickeya fangzhongdai, the causal agent of bleeding canker of pear, is a new member of the Dickeya genus and the only one that infects woody plants. Recent studies have reclassified several Dickeya isolates as D. fangzhongdai, which were isolated from various environments, including water, Phalaenopsis sp. and Aglaonema sp. To provide genomic characterization of D. fangzhongdai isolates from pear, the genomes of D. fangzhongdai strain JS5(=China General Microbiological Culture Collection Center, CGMCC 1.15464 ~T=DSM 101947 ~T), along with two other isolates, LN1 and QZH3, were sequenced and compared to those of other Dickeya spp. Homology greater than 99% was observed among three D. fangzhongdai strains. Plasmid, type IV secretion system(T4 SS) and type IV pili(TFPs) were found in genomes of D. fangzhongdai isolates. Comparative analysis of the type Ⅲ secretion systems(T3 SS), type Ⅲ secretion effectors(T3 SE), plant cell wall degradation enzymes(PCWDE) and membrane transport proteins of Dickeya spp. showed some differences which might reflect the variations of virulence, phylogenetic and phenotypic characteristics of Dickeya spp. In addition, deletion mutant of TFP in D. fangzhongdai JS5 showed no twitching motility and reduced virulence and biofilm formation. The fingdings of the distinctive plasmid, T4 SS and TFPs, as well as the differences of T3 SE, PCWDE and membrane transport proteins make D. fangzhongdai isolates unique. These results also suggested that acquisition of virulence genes by horizontal gene transfer might play some role in the genetic variation of D. fangzhongdai.

Real-time PCR assay for detection of Dickeya fangzhongdai causing bleeding canker of pear disease in China

Bleeding canker, caused by Dickeya fangzhongdai, is a devastating disease of pear in China. The bacterium causes cankers, branch die-back, and eventually kills pear trees. The typical sign of bleeding canker infection is a rusty-brown bacterial ooze that exudes down from cankers onto branches or trunks. However, early symptoms and signs are inconspicuous, which makes effective disease management difficult. Detection and identification of D. fangzhongdai are time-consuming and difficult because no rapid method exists to date. In this study, a Taq Man real-time PCR assay was developed for D. fangzhongdai based on an elongation factor G(fus A) gene. The real-time PCR assay detected 0.2 pg μL^-1 DNA and 1×10^3 Cfu m L^-1 of D. fangzhongdai. Based on this assay, bleeding canker on asymptomatic pear trees can be diagnosed as early as 5 days after infection. The real-time PCR assay can facilitate disease management by providing early and accurate diagnosis of the bleeding canker disease of pear.

Cloning and Bioinformatics Analysis of the GlROP6 gene in Glehnia littoralis

Rho-related GTPase from plants(ROP)proteins play an essential role in plant stress resistance.In this study,the full-length GlROP6 gene was cloned based on G.littoralis transcriptome sequencing data acquired in response to salt stress.The protein sequence,conserved domains,secondary structure,three-dimensional structure,phylogenetic relationships,and expression pattern of the GlROP6 gene were systematically analysed.Our results showed that the full-length GlROP6 gene had an open reading frame of 606 bp,which encoded 201 amino acid residues with a relative molecular weight of 22.23463 kDa and a theoretical isoelectric point of 9.06.Amino acid sequence analyses indicated that the structure of the GlROP6 protein was conserved,and included five G-box motifs(G1–G5),an effector binding region,a Rho insert region and a C-terminal hypervariable region.According to our phylogenetic analysis,the GlROP6 protein was closely related to the ROP protein of Daucus carota subsp.Sativus.Our quantitative real-time PCR results revealed that GlROP6 was highly expressed in flower,and GlROP6 expression was significantly upregulated in G.littoralis roots treated with NaCl.This study will facilitate investigations into the function of GlROP genes in response to salt stress in G.littoralis.

A Preliminary Study on Cold Tolerance in Cinnamomum japonicum Sieb. at Different Ages

[ Objective] This study aimed to further expand the production and application of Cirmamomumjaponicum Sieb. and investigate the mechanism of cold resistance in evergreen broad-leaved tree species. [ Method] Using adult Cinnamonum campora as control, during the entire wintering period from October 2011 to March 2012, the wintering adaptability of C. japonicum leaves at different ages was observed; the electrolyte leakage was measured under indoor low-temperature treatment to calculate the semi-lethal temperature （LT50） by fitting Logistic equation, thus conducting dynamic analysis and comprehensive evaluation of the cold re- sistance in C. japonicum. [ Result] The results showed that the chilling injuries in leaves of different materials were aggravated with the decreasing temperature. According to the results of five times of dynamic observation on wintering adaptability, the cold resistance in different materials presented a decreasing order of adult C. japonicum medium-aged C. japonicum 〉 young C. japon/cum 〉 adult C. campora. The relationship between indoor low temperature and leaf injury rate showed an S-shaped curve. The significance test suggested relatively high fitting degree with Logistic equation. The semi-lethal temperature presented a V-shaped curve with the changing temperature and roached the minimum during late December to early February in the following year. The dynamic changes and minimum value of semi-lethal temperature indicated a consistent order of cold resistance in different materials to the results of wintering adaptability observation. Therefore, results of two analysis methods could be combined for cold resistance evaluation. [ Conclusion] This study laid the foundation for the introduction and populariza- tion of C. japonicum in northern gardens along the Yangtze River.

Diversity Assessment of an Endemic Carpinus oblongifolia(Betulaceae)Using Specific-Locus Amplified Fragment Sequencing and Implications for Conservation

Carpinus oblongifolia is an endemic species and the extant wild populations show a fragmentation distribution in the Baohua Mountain of Jiangsu Province in eastern China.Understanding of genetic diversity plays an important role in C.oblongifolia survival and sustainable development.The wild C.oblongifolia population was artificially divided into four subpopulations according to the microhabitats,and another two subpopulations were constructed by progeny seedlings cultivated with the mature seeds.Then,the leaf buds of 80 individuals from six subpopulations were sampled to develop single nucleotide polymorphisms(SNPs)using specific-locus amplified fragment sequencing(SLAF-seq).Based on these SNPs,we aimed to characterize the genetic diversity and population structure of C.oblongifolia and provide an illumination and reference for effective management of such a small endemic population.The level of genetic diversity was low at the species level,and the progeny subpopulations had a relatively higher genetic diversity than the wild subpopulations.This may be attributed to a high gene flow and an excess heterozygosity to reduce the threat of genetic drift-based hazards.Moreover,the progeny subpopulations had the ability to form new clusters and a great contribution to the genetic structure variation of C.oblongifolia.These results will assist with the development of conservation and management strategies,such as properly evacuating competitive trees to provide more chance for pollen and seed flow in situ conservation,and establishing sufficient seedling plantlets under laboratory conditions for reintroduction to enlarge the effective population size.

Electrochemical Identification of Yulania spp. by Fingerprinting of Leaves Using Glassy Carbon Electrode

In this communication,we used electrochemical sensor for recording the electrochemical profiles of eleven species of Yulania spp.from leaf extract.Two solvents and two buffer conditions were used for electrochemical fingerprints collection.Their electrochemical fingerprints can be converted to different patterns and consequently for species recognition.The results indicate the pattern recognition is much convenient than that of the recognition of species directly using voltammetric signal.The current information in electrochemical fingerprinting represents the type and amount of electrochemically active molecules,which linked to the genetic differences among the plants.Therefore,the electrochemical fingerprints were applied for further phylogenetic study.The phylogenetic tree deduced from voltametric curves is divided into three main groups.The first clade contains Y.denudate,Liriodendron chinense,Y.cylindrica,Y.biondii,Y.sprengeri.The second clade contains Y.zenii,Y.liliiflora,Y.kobus,and Y.amoena.The third clade contains Y.×soulangeana,Manglietia fordiana and Y.sinostellata.In addition,Y.salicifolia is not in these main clades.The results demonstrate that electrochemical fingerprinting can be used as a com-plementary tool in the study of phylogenetics.

Transcriptome analysis of Clematis lanuginosa: Novel features of the molecular events occurring under heat-shock stress

The weak heat tolerance of Clematis ornamental varieties negatively affects their ornamental qualities in the summer.To elucidate heat resistance mechanisms,Clematis lanuginosa,which is an important original parent of the Clematis large-flowered group of ornamental varieties,was selected for use in this study.Here,six libraries,including three biological replicates each of control and heat-shock stress samples,were determined using RNA-sequencing technology.In total,62,050 unigenes were obtained,and 6,439 unigenes exceeded 1 kb in length.A total of 42,377 unigenes were annotated using six databases.Between the two treatments,2,165 differentially expressed genes were identified,with 1,565 being up-regulated and 600 down-regulated.In addition,51 heat-shock protein-encoding genes were identified,among which the small heat-shock proteins accounted for 68.63%.In total,two heat-shock factors and 12 ribosomal proteins were significantly up-regulated under heatstress conditions.The differential expressions of ethylene-responsive transcription factor,chalcone synthase,cysteine-rich receptor-like kinase and aspartic protease unigenes in guard cells were induced by heat-shock.The data obtained will assist the elucidation of the molecular events underlying heat-stress responses in C.lanuginosa.

Genetic resources of lotus (Nelumbo) and their improvement

Lotus(Nelumbo)is one of the top ten flowers in China,which has high ornamental,edible and medicinal value.Lotus has a been cultivated for thousands of years.Through discovery and cultivation,more than 4,000 cultivars have been recorded.However,the information related to lotus breeding is quite scattered,and the related genetic rules and trait formation mechanisms are still poorly understood,which has caused a greater impact on lotus genetic breeding.This article systematically introduces lotus germplasm resources,including wild species and cultivated species,summarizes lotus breeding methods and breeding directions,and focuses on the latest progress in the isolation and functional identification of structural and regulatory genes related to important horticultural traits.Prospects for the protection and utilization of lotus resources,breeding and industrialization are reported.