Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants

Endogenous auxin is an important regulator of in vivo organ development,but its role in in vitro organogenesis is unclear.It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end.Here,we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis.Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene,we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport.Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis.An auxin transport inhibitor,N-1-naphthylphthalamic acid(NPA),can also enhance shoot organogenesis independent of its action on polar auxin transport.Finally,we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent.Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport,as well as auxin–cytokinin interactions,in in vitro shoot organogenesis.Meanwhile,our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.

Preparation of Monoclonal Antibody Against PthA-NLS and Cloning of the Relative ScFv Gene

The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.

Vitamin B3 inhibits apoptosis and promotes autophagy of isletβcells under high glucose stress

Background:Hyperglycemia is a typical symptom of diabetes.High glucose induces apoptosis of isletβcells.While autophagy functions in cytoprotection and autophagic cell death.The interaction between autophagy and apoptosis is important in the modulation of the function of isletβcells.Vitamin B3 can induce autophagy and inhibit isletβapoptosis.Method:The mechanism of vitamin B3-mediated protective effect on the function of isletβcells was explored by the method of western blot,immunofluorescence and flow cytometry.Results:In the present study,high glucose stress increased the apoptosis rate,while vitamin B3 reduced the apoptosis rate.The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated.Vitamin B3 increased the number of autophagosomes and increased the light chain(LC)3-II/LC3-I ratio.In contrast,vitamin B3 decreased sequestosome 1(SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinaseβ-1(p70S6K/S6K1),which was a substrate of mammalian target of rapamycin(mTOR)under normal and high glucose stress.To further verify the protective effect of vitamin B3 on apoptosis,we treated isletβcell RIN-m5F with autophagy inhibitor 3-methyladenine(3-MA).Vitamin B3 decreased the apoptosis rate under high glucose stress,while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion:Our data suggested that vitamin B3 reduced the apoptosis rate ofβcells,possibly through inducing autophagy under high glucose stress.

Mapping and predicting a candidate gene for flesh color in watermelon

The color of watermelon flesh is an important trait determined by a series of carotenoids.Herein,we used Cream of Saskatchewan(pale yellow flesh)and PI 186490(white flesh)as parental materials for an F2 segregation and initial mapping using the bulked segregant analysis sequencing(BSA-seq)strategy.The BSA results revealed a flesh colorrelated QTL that spans approximately 2.45 Mb on chromosome 6.This region was preliminarily positioned in a 382-kb segment,and then narrowed down into a 66.8-kb segment with 1260 F2 individuals.A total of nine candidate genes were in the fine mapping interval,but only Cla007528(encoding chlorophyllase)had non-synonymous mutations and was significantly expressed between the parental materials throughout flesh development.We also checked the expression patterns of the carotenoid metabolic pathway genes based on RNA-seq data and qRT-PCR validation.Three genes in the xanthophyll cycle(ClCHYB,ClNCED-1 and ClNCED-7)exhibited differential expression patterns between the two parental lines at different flesh color formation stages.ClPSY1,ClPDS,ClZDS,ClCHXE,ClCRTISO and ClLCYB also exhibited clearly different expression patterns accompanied by carotenoid accumulation.

Effect of Water Deficit Stress on Isotope ^15N Uptake and Nitrogen Metabolism of Newhall Orange and Yamasitaka Mandarin Seedling

Soil water content significantly influenced uptake and distribution of ^15N in both Newhall and Yamasitaka. The content of ^15N uptake in treated plants was less than that in controlled plants, under 20% soil water content, ^15N was only taken up 16.02% by Newhall and 10.11% by Yamasitaka. The most ^15N was detained in root and old shoots under water stress. Protein concentration in two cultivars significantly decreased by water deficit stress, protein content of Newhall and Yamasitaka in controlled plants was 16.29 mg/g fresh weight and 15.89 mg/g fresh weight, but at 20% of water content, these were 9.60 mg/g fresh weight and 9.02 mg/g fresh weight. Water stress increased concentration of NH3-NH4^＋, Arginine and Proline. Compared with control plants, concentrations of NH3-NH4^＋ in both Newhall and Yamasitaka at 20% water content treatment increased 5.83 fold and 5.71 fold, Arginine increased 197% and 205%, and Proline increased 112% and 132%.

Metabolic Profiling in Banana Pseudo-Stem Reveals a Diverse Set of Bioactive Compounds with Potential Nutritional and Industrial Applications

Banana(Musa spp.)is an ancient and popular fruit plant with highly nutritious fruit.The pseudo-stem of banana represents on average 75%of the total dry mass but its valorization as a nutritional and industrial by-product is limited.Recent advances in metabolomics have paved the way to understand and evaluate the presence of diverse sets of metabolites in different plant parts.This study aimed at exploring the diversity of primary and secondary metabolites in the banana pseudo-stem.Hereby,we identified and quantified 373 metabolites from a diverse range of classes including,alkaloids,flavonoids,lipids,phenolic acids,amino acids and its derivatives,nucleotide and its derivatives,organic acids,lignans and coumarins,tannins,and terpene using the widely-targeted metabolomics approach.Banana pseudo-stem is enriched in metabolites for utilization in the food industry(L-lysine and L-tryptophan,L-glutamic acid,Phenylalanine,Palmitoleic acid,α-Linolenic acid,and Lauric acid,and Adenine)and pharmaceutical industry(Guanosine and Cimidahurinine,Bergapten,Coumarins,Procyanidin A2,Procyanidin B1,Procyanidin B3,Procyanidin B2,and Procyanidin B4,Asiatic acid).The metabolome of banana pseudo-stem with integration across multiomics data may provide the opportunity to exploit the rich metabolome of banana pseudo-stem for industrial and nutritional applications.

Molecular Detection and Characterization of Citrus Viroids

Total RNA was isolated from the phloem of young shoots and retro-transcripted to cDNA by reverse transcriptasepolymerase chain reaction （RT-PCR） with specific primers; the amplified cDNA fragments were sequenced for identification of subtypes. In this study, an effective RT-PCR technique was established for detecting citrus viroids, and three citrus viroids were recognized, namely, CEVd, CVd-II, and CVd-III. The latter two were identified for the first time in China. The sequences of CVd-Ⅱ were different from all the three subtypes reported in the GenBank and seemed to be a new subtype. CVd-Ⅲb detected in the present study could be further developed as a dwarfing agent.

Induction and Identification of Polyploidy Plants from Superior Individuals of Wild Lonicera edulis Turcz. in Changbai Mountains

[ Objectives] This study was conducted to provide high-quality polyploid for returning the grain plots to forestry and promote the rapid development of Lonicera edulis Turcz. [ Methods] With superior individuals of wild L. edulis in Changbai Mountains as experimental materials and colchicine as inducer, polyploid induction was performed twice on diploid plants by addition method, and 7 polyploidy plants were identified from 11 regenerated plantlets. [Results] Among the 7 polyploidy plants, four materials were tetraploid, the concentration and treatment time of which were 300 mg/L and 7 and 14 d, respectively; and three materials were octaploid, the concentrations and treatment time of which were 300 and 500 mg/L and 14 d, respectively. [ Conclusions] The results of this study showed that colchicine had a higher polyploid induction rate for L. edulis with lower toxic and side effects, and the polyploidy plants could restore to normal growth state after several times of cubculture. Therefore, colchicine is an polyploid inducer suitable for L. edulis.

Investigating the Effectiveness of Road-related Mitigation Measures under Semi-controlled Conditions:A Case Study on Asian Amphibians

Road traffic is the main factor causing the decline in amphibian populations worldwide. The proper design of an amphibian tunnel is one of the most efficient measures to mitigate the negative impacts of road traffic on amphibians. However, no study has investigated the effectiveness of amphibian tunnels under semi-controlled conditions in Asian amphibians. Here, we selected two representative amphibian species, the Chinese brown frog, Rana chensinensis, and the Asiatic toad, Bufo gargarizans, which suffer the most severe road mortality along the roads in Northeast China. We placed experimental arrays of culverts of various sizes(diameters of 1.5, 1, and 0.5 m for circular culverts; side lengths of 1.5, 1, and 0.5 m for box culverts), and substrate type(soil, concrete, and metal) to examine the preferences of both species during the migratory season between May and September in 2016 and 2017. The results revealed that the Chinese brown frog preferred mid-and large-sized culverts as well as soil culverts. We concluded that culverts with a side length ≥ 1 m, lined with soil, and accompanied by a ≥ 0.4 m high guide drift fence and ≤ 45° gradient on the roadside ditch wall would best facilitate road crossings for both species and likely for other amphibian species in Northeast China.

Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

Introgression of High Yield Genes from Lycopersicon hirsutum acc.LA1777 Using CAPS Marker

The idea behind this study is to show that using high yield genes from a wild tomato can enrich tomato breeding resources and accelerate tomato breeding programs. In this study, the near-isogenic line TA1229 containing a 24-cM introgression at the bottom of chromosome 1 from Lycopersicon acc. LA1777, affects several higher yield traits. The TA1229×9706 BC1 population was analyzed by marker-assisted selection and the traits of the population were evaluated. Twenty-three recombinant individuals that carried a shorter segment than TA1229 were obtained. Among them, 16 lines with the chromosome 1 recombinant segment can increase tomato yield and a QTL affecting yield was found between TG53 and TG158. Sixteen recombinant lines are useful to improve the tomato variety.

Study on Pollination Compatibility of Superior Lonicera caerulea Individuals

In order to solve the problems including low fruit-bearing rate and low yield caused by blind plantation in production, the medium formula for pollen germination of Lonicera caerulea L. var. edulis Turcz. ex Herd., its pollination habit, self-compatibility and open pollination compatibility between different superior individuals were investigated with superior individuals from superior wild individuals of L. caerulea in Changbai Mountains as experimental materials. Self-compatibility and open pollination compatibility among different fine plants provide theoretical basis for its further application and further selection of new varieties. The results showed the optimal medium for germination of L. caerulea pollen was 27.5% sucrose ＋ 100 PPM boric acid with a pH value of 6.0. On this medium, the superior individuals L1, L2 and L3 had the pollen germination rates of 53.4%, 50.9% and 51.6%, respectively. The three superior individuals had no significant differences in the quantity of germinated pollen tubes, which ranged from 1 to 4. The three excellent single plants were the most likely to germinate a single pollen tube, accounting for 83.3% of the pollen germinated and 89.6% of the pollen germinated. Therefore, in production, it is necessary to plant L1 with a certain amount of pollination trees or plant it together with multiple species, to improve its yield. On the contrary, L2 and L3 have no need for pollination trees theoretically, but whether the fruits obtained by self-pollination and outcrossing differ in quality still needs further study.

The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism

The overuse of antibiotics in animal agriculture and medicine has caused a series of potential threats to public health. Macleaya cordata is a medicinal plant species from the Papaveraceae family, providing a safe resource for the manufacture of antimicrobial feed additive for livestock. The active constituents from M. cordata are known to include benzylisoquinoline alkaloids （BIAs） such as sanguinarine （SAN） and chelerythrine （CHE）, but their metabolic pathways have yet to be studied in this non-model plant. The active biosynthesis of SAN and CHE in M. cordata was first examined and confirmed by feeding ^13C-labeled tyrosine. To gain further insights, we de novo sequenced the whole genome of M. cordata, the first to be sequenced from the Papaveraceae family. The M. cordata genome covering 378 Mb encodes 22,328 predicted protein-coding genes with 43.5% being transposable elements. As a member of basal eudicot, M. cordata genome lacks the paleohexaploidy event that occurred in almost all eudicots. From the genomics data, a complete set of 16 metabolic genes for SAN and CHE biosynthesis was retrieved, and 14 of their biochemical activities were validated. These genomics and metabolic data show the conserved BIA metabolic pathways in M. cordata and provide the knowledge foundation for future productions of SAN and CHE by crop improvement or microbial pathway reconstruction.