Comamonas endophytica sp. nov., a novel indole acetic acid producing endophyte isolated from bamboo in China.

Two novel indole acetic acid-producing strains, 5MLIRT and D4N7, were isolated from Indosasa shibataeoides in Yongzhou, Hunan province, and Phyllostachys edulis in Hangzhou, Zhejiang province, respectively. Based on their 16S rRNA sequences, strains 5MLIRT and D4N7 were closely related to Comamonas antarcticus 16-35-5T (98.4 % sequence similarity), and the results of 92-core gene phylogenetic trees showed that strains 5MLIRT and D4N7 formed a phylogenetic lineage within the clade comprising Comamonas species. The complete genome size of strain 5MLIRT was 4.49 Mb including two plasmids, and the DNA G+C content was 66.5 mol%. The draft genome of strain D4N7 was 4.26 Mb with 66.7 mol% G+C content. The average nucleotide identity and digital DNA-DNA hybridization values among strain 5MLIRT and species in the genus Comamonas were all below the species delineation threshold. The colonies of strain 5MLIRT and D4N7 were circular with regular margins, convex, pale yellow and 1.0-2.0 mm in diameter when incubated at 30 °C for 3 days. Strains 5MLIRT and D4N7 grew optimally at 30 °C, pH 7.0 and 1.0 % NaCl. The respiratory isoprenoid quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Polyphasic analyses indicated that strains 5MLIRT and D4N7 could be distinguished from related validly named Comamonas species and represent a novel species of the genus Comamonas, for which the name Comamonas endophytica sp. nov. is proposed. The type strain is 5MLIRT (=ACCC 62069T=GDMCC 1.2958T=JCM 35331T).

Characterization of color variation in bamboo sheath of Chimonobambusa hejiangensis by UPLC-ESI-MS/MS and RNA sequencing.

BACKGROUND: Chimonobambusa hejiangensis (C.hejiangensis) is a high-quality bamboo species native to China, known for its shoots that are a popular nutritional food. Three C.hejiangensis cultivars exhibit unique color variation in their shoot sheaths, however, the molecular mechanism behind this color change remains unclear. METHODS: We investigated flavonoid accumulation in the three bamboo cultivar sheaths using metabolomics and transcriptomics. RESULTS: UPLC-MS/MS identified 969 metabolites, with 187, 103, and 132 having differential accumulation in the yellow-sheath (YShe) vs. spot-sheath (SShe)/black-sheath (BShe) and SShe vs. BShe comparison groups. Flavonoids were the major metabolites that determined bamboo sheath color through differential accumulation of metabolites (DAMs) analysis. Additionally, there were 33 significantly differentially expressed flavonoid structural genes involved in the anthocyanin synthesis pathway based on transcriptome data. We conducted a KEGG analysis on DEGs and DAMs, revealing significant enrichment of phenylpropanoid and flavonoid biosynthetic pathways. Using gene co-expression network analysis, we identified nine structural genes and 29 transcription factors strongly linked to anthocyanin biosynthesis. CONCLUSION: We identified a comprehensive regulatory network for flavonoid biosynthesis which should improve our comprehension of the molecular mechanisms responsible for color variation and flavonoid biosynthesis in bamboo sheaths.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Massilia phyllostachyos sp. nov., Isolated from the Roots of Moso Bamboo in China.

A Gram-negative, strictly aerobic, motile, and rod-shaped bacterial strain G4R7T was isolated from the roots of moso bamboo (Phyllostachys edulis) in Zhejiang, Hangzhou Province, China. After comparing 16S rRNA gene sequences, strain G4R7T exhibited the highest similarities with Massilia neuiana PTW21T (98.3%), followed by M. agri K-3-1T (98.3%), M. consociate CCUG 58010T (97.7%), M. niastensis 5516S-1T (97.7%) and M. yuzhufengensis ZD1-4T (97.6%). The phylogenetic analysis revealed that strain G4R7T belonged to the genus Massilia. The draft genome of strain G4R7T was 5.81 Mb, and the G+C content was 64.4%. The average nucleotide identity values between G4R7T and another related member of the genus Massilia ranged from 76.6 to 87.2%, and the digital DNA-DNA hybridization ranged from 20.7 to 27.9%. Strain G4R7T grew at 15-37 °C (optimum 25-30 °C) and pH 6.0-9.0 (optimum pH 7.0) in the presence of 0-3% (w/v) NaCl (optimum 0%). The respiratory quinone was Q-8, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid. The major cellular fatty acids were C10:0 3OH, C12:0, C12:0 2OH, and C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). As per the data from chemotaxonomic, phylogenetic, and phenotypic evidence, strain G4R7T represents a new species of genus Massilia, for which the name Massilia phyllostachyos sp. nov. is proposed. The type strain is G4R7T (=ACCC 61911T=GDMCC 1.2961T=JCM 35225T).

Comparative Analyses of Anatomical Structure, Phytohormone Levels, and Gene Expression Profiles Reveal Potential Dwarfing Mechanisms in Shengyin Bamboo (Phyllostachys edulis f. tubaeformis).

Moso bamboo (Phyllostachys edulis) is one of the most important bamboo species in China and the third most important plant species for timber production. However, the dwarf variant of moso bamboo, P. edulis f. tubaeformis (shengyin bamboo), which has shortened internodes, is not well studied. We used anatomical, hormonal, and transcriptomic approaches to study internode shortening and shoot growth in dwarf shengyin and wild moso bamboo. Phenotypic and anatomical observations showed that dwarfing in shengyin bamboo is due to reduced internode length, and the culm fibers in shengyin bamboo are significantly shorter and thicker than in wild moso bamboo. We measured the levels of endogenous hormones in the internodes and found that shengyin bamboo had lower levels of four hormones while two others were higher in wild moso bamboo. Comparative transcriptome analyses revealed a potential regulating mechanism for internode length involving genes for cell wall loosening-related enzymes and the cellulose and lignin biosynthesis pathways. Genes involved in hormone biosynthesis and signal transduction, especially those that showed significant differential expression in the internodes between shengyin and wild moso bamboo, may be important in determining the shortened internode phenotype. A hypothesis involving possible cross-talk between phytohormone signaling cues and cell wall expansion leading to dwarfism in shengyin bamboo is proposed. The results presented here provide a comprehensive exploration of the biological mechanisms that determine internode shortening in moso bamboo.

BeMADS1 is a key to delivery MADSs into nucleus in reproductive tissues-De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development.

BACKGROUND: The bamboo Bambusa edulis has a long juvenile phase in situ, but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by use of two sequencing platforms: Illumina and 454. Combination of the two datasets resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes control flower development, and the ABCDE model has been developed to explain how the genes function together to create the different whorls within a flower. RESULTS: As a case study, published floral development-related OsMADS proteins from rice were used to search the B. edulis transcriptome datasets, identifying 16 B. edulis MADS (BeMADS). The BeMADS gene expression levels were determined qRT-PCR and in situ hybridization. Most BeMADS genes were highly expressed in flowers, with the exception of BeMADS34. The expression patterns of these genes were most similar to the rice homologs, except BeMADS18 and BeMADS34, and were highly similar to the floral development ABCDE model in rice. Transient expression of MADS-GFP proteins showed that only BeMADS1 entered leaf nucleus. BeMADS18, BeMADS4, and BeMADS1 were located in the lemma nucleus. When co-transformed with BeMADS1, BeMADS15, 16, 13, 21, 6, and 7 translocated to nucleus in lemmas, indicating that BeMADS1 is a key factor for subcellular localization of other BeMADS. CONCLUSION: Our study provides abundant B. edulis transcriptome data and offers comprehensive sequence resources. The results, molecular materials and overall strategy reported here can be used for future gene identification and for further reproductive studies in the economically important crop of bamboo.

Chimonobambusafarcta (Poaceae, Bambusoideae), a new species from western Guangxi, China with taxonomic notes on C.pubescens and C.luzhiensis.

Chimonobambusafarcta, a new species of temperate woody bamboos from western Guangxi, China is described and illustrated. The new species is similar to C.pubescens in the solid internodes of culms, but differs in having taller culm to 7 m with longer verrucose internodes to 23.5 cm and intranodes to 4 mm, intranode usually with a ring of 7-9 root thorns below mid-culm, abaxially brown or brown-purple verrucose-setose culm leaf sheaths with indistinct transverse veins, conspicuously developed culm leaf blades to 3.2 cm long, longer foliage leaf sheaths to 5.2 cm, larger and broader foliage leaf blades to 22 × 1.4 cm. It also somewhat resembles C.convoluta, but can be easily distinguished by having solid internodes and longer intranode 2-4 mm, very prominent nodes with supranodal ridge obviously more elevated than sheath scar, usually persistent and sometimes brownish striate culm leaf sheaths, longer culm leaf blades to 3.2 cm, and abaxially glabrous foliage leaf sheaths. Based on the morphological characteristics, this new species is assigned to C.sect.Chimonobambusa. The character description of C.pubescens are revised for its culm to 2.1 m tall, 1.1 cm in diameter and glabrous foliage leaf blades. The systematic position of C.luzhiensis is discussed, and this species is proposed as a member of C.sect.Chimonobambusa.

Chimonobambusapleiacantha (Poaceae, Bambusoideae), a new species from southeast Sichuan, China.

Chimonobambusapleiacantha, a newly-identified species of the genus Chimonobambusa Makino from southeast Sichuan, China, is meticulously described and illustrated herein. It is morphologically similar to Ch.tuberculata, but differs in having 4-angled internodes, thicker wall to 4.5-8.5 mm, more reclinate and elongated root thorns to 5-8 mm long, culm internodes with three grooves and two longitudinal ridges on the branch-bearing side, persistent culm leaves, densely brown-purple setose at the bottom of culm leaf sheaths together with sheath scar, developed foliage leaf fimbriae, 6-8 on each shoulder, ca. 3-8 mm long, abaxially white pubescent foliage leaf blades. Phenologically, new shoots usually appear in September to October. In the light of these key morphological and phenological characteristics, Ch.pleiacantha was identified as a new species of the genus Ch. Makino which is different from published species of this genus.

The complete chloroplast genome of Phyllostachys edulis f. tubiformis (Bambusoideae): a highly appreciated type of ornamental bamboo in China.

Phyllostachys edulis (Carr.) H. de Lehaie f. tubiformis (S.Y.Wang) Ohrnberger (shengyin bamboo in Chinese) is a dwarf form of moso bamboo, which has important ornamental value. In this study, the complete chloroplast genome of P. edulis f. tubiformis was reported. The complete chloroplast genome of P. edulis f. tubiformis is a double-circular DNA of 139,678 bp in length with 38.89% G + C content, and contains 126 genes, including 84 protein-coding genes, eight ribosomal RNA (rRNA) genes, and 34 transfer RNA (tRNA) genes. Phylogenetic analysis results strongly supported that P. edulis f. tubiformis was clustered with the other infra-species of P. edulis, although its morphology is quite different from moso bamboo.

Heavy metals in bamboo shoots from Southeastern China and risk assessment.

Bamboo shoot is an indispensable vegetable in Southeastern China, so a survey of heavy metals in bamboo shoots is a relevant topic of interest. The present study sought to analyse the content of seven heavy metals (As, Cd, Cr, Cu, Mn, Pb and Zn) in six bamboo shoot species from Southeastern China. Percentages of 3.8% (Cd) and 8.9% (Pb) of the samples exceeded the maximum limits as established by the Chinese legislation. Further health risk assessment exhibited that the Cd intake contribution of these samples could not be negligible. The hazard index (HI) at average exposure was less than one. The average HI values in moso bamboo shoot were significantly higher than those of other bamboo shoot species. Overall, the results indicated that the intake of bamboo shoots is safe for consumers in Southeastern China.

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.

The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

Flowering of Woody Bamboo in Tissue Culture Systems.

Flowering and subsequent seed set are not only normal activities in the life of most plants, but constitute the very reason for their existence. Woody bamboos can take a long time to flower, even over 100 years. This makes it difficult to breed bamboo, since flowering time cannot be predicted and passing through each generation takes too long. Another unique characteristic of woody bamboo is that a bamboo stand will often flower synchronously, both disrupting the supply chain within the bamboo industry and affecting local ecology. Therefore, an understanding of the mechanism that initiates bamboo flowering is important not only for biology research, but also for the bamboo industry. Induction of flowering in vitro is an effective way to both shorten the flowering period and control the flowering time, and has been shown for several species of bamboo. The use of controlled tissue culture systems allows investigation into the mechanism of bamboo flowering and facilitates selective breeding. Here, after a brief introduction of flowering in bamboo, we review the research on in vitro flowering of bamboo, including our current understanding of the effects of plant growth regulators and medium components on flower induction and how in vitro bamboo flowers can be used in research.

Correlation between DNA methylation and chronological age of Moso bamboo (Phyllostachys heterocycla var. pubescens).

BACKGROUND: Chronological age is the primary consideration when studying the physiological development, aging, and flowering of bamboo. However, it's difficult to determine bamboo's chronological age if the time of germination is unknown. To investigate the chronological age of bamboo from the genomic DNA methylation profile, methylation-sensitive amplification polymorphism (MSAP) was employed to analyze the genomic DNA methylation of Moso bamboo (Phyllostachys heterocycla var. pubescens) from stands of nine germination-ages, using six primer pairs which have previously been shown to yield methylation rates that reflect the age of Moso bamboo. RESULTS: The results showed that the total genomic DNA methylation rates in Moso bamboo at different chronological ages were significantly different, and the increase in genomic DNA methylation rate was consistent with the increase of chronological age. Six primer pairs displayed different genomic DNA methylation rates in Moso bamboo of nine age's group; however, a significantly positive correlation existed among these primer pairs. An integrated index was obtained by performing principal component analysis on the six primer pairs to represent the genomic DNA methylation levels in Moso bamboo of various chronological ages, and a quadratic curve between the chronological age and genomic DNA methylation levels was obtained. CONCLUSIONS: Such a relationship between DNA methylation and its chronological age may serve a reference for its aging study in Moso bamboo.

Protocol for callus induction and somatic embryogenesis in Moso Bamboo.

Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10-20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5-2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0-7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0-7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.

Ternary oxovanadium(IV) complexes with amino acid-Schiff base and polypyridyl derivatives: synthesis, characterization, and protein tyrosine phosphatase 1B inhibition.

To investigate the structure-activity relationship of vanadium complexes in inhibiting protein tyrosine phosphatase1B (PTP1B), eight mixed-ligand oxovanadium(IV) complexes, [V(IV)O(SalAla)(NN)] (H(2)SalAla for salicylidene alanine, NN for N,N'-donor heterocyclic base, namely, 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4)), [V(IV)O(SalLys)(dpq)] (5), [V(IV)O(SalLys)(dppz)] (6), [V(IV)O(SalAsp)(dppz)], (7) and [V(IV)O(SalTrp)(dppz)] (8)), of which 3-8 are new, have been prepared and characterized by elemental analysis, infrared, UV-visible, electrospray ionization mass spectrometry and conductivity. The molar conductance data confirmed the non-electrolytic nature of the complexes in DMSO solution. The coordination in [V(IV)O (SalAla)(phen)] (2) was confirmed by X-ray crystal structure analysis. The oxidation state of V(IV) with d(1) configuration in 2 was confirmed by EPR. The speciation of VO-SalAla-phen in aqueous solution was investigated by potentiometric pH titrations. The results indicate that the main species are two ternary complexes at the pH range 7.0-7.4. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of PTP1B with IC(50) values in the range of 62-597nM, approximately 3-10 fold weaker in potency than those of similar mixed-ligand oxovanadium(IV) complexes of salicylidene anthranilic acid (SAA) derivative with polypyridyl ligands, except complex 8, which exhibits comparable or better inhibition activity than those of the mixed-ligand oxovanadium(IV) complexes of SAA derivative with polypyridyl ligands. The results demonstrate that the structures of vanadium complexes influence the PTP1B inhibition activity. Kinetics assays reveal that complex 2 inhibits PTP1B in a competitive manner.