Transcriptional and protein structural characterization of homogentisate phytyltransferase genes in barley, wheat, and oat.

BACKGROUND: Homogentisate phytyltransferase (HPT) is the critical enzyme for the biosynthesis of tocopherols (vitamin E), which are the major lipid-soluble antioxidants and help plants adapt to various stress conditions. HPT is generally strictly conserved in various plant genomes; however, a divergent lineage HPT2 was identified recently in some Triticeae species. The molecular function and transcriptional profiles of HPT2 remain to be characterized. RESULTS: In this study, we performed comprehensive transcriptome data mining of HPT1 and HPT2 in different tissues and stages of barley (Hordeum vulgare), wheat (Triticum aestivum), and oat (Avena sativa), followed by qRT-PCR experiments on HPT1 and HPT2 in different tissues of barley and wheat. We found that the common HPT1 genes (HvHPT1, TaHPT1s, and AsHPT1s) displayed a conserved transcriptional pattern in the three target species and were universally transcribed in various tissues, with a notable preference in leaf. In contrast, HPT2 genes (HvHPT2, TaHPT2, and AsHPT2) were specifically transcribed in spike (developmentally up-regulated) and shoot apex tissues, displaying a divergent tissue-specific pattern. Cis-regulatory elements prediction in the promoter region identified common factors related to light-, plant hormone-, low temperature-, drought- and defense- responses in both HPT1s and HPT2s. We observed the transcriptional up-regulation of HvHPT1 and HvHPT2 under various stress conditions, supporting their conserved function in environmental adaption. We detected a clear, relaxed selection pressure in the HPT2 lineage, consistent with the predicted evolution pattern following gene duplication. Protein structural modelling and substrate docking analyses identified putative catalytic amino acid residues for HvHPT1 and HvHPT2, which are strictly conserved and consistent with their function in vitamin E biosynthesis. CONCLUSIONS: We confirmed the presence of two lineages of HPT in Triticeae and Aveninae, including hexaploid oat, and characterized their transcriptional profiles based on transcriptome and qRT-PCR data. HPT1s were ubiquitously transcribed in various tissues, whilst HPT2s were highly expressed in specific stages and tissue. The active transcription of HPT2s, together with its conserved cis-elements and protein structural features, support HPT2s' role in tocopherol production in Triticeae. This study is the first protein structural analysis on the membrane-bound plant HPTs and provides valuable insights into its catalytic mechanism.

Investigating microRNA-mediated regulation of the nascent nuclear transcripts in plants: a bioinformatics workflow.

Most of the microRNAs (miRNAs) play their regulatory roles through posttranscriptional target decay or translational inhibition. For both plants and animals, these regulatory events were previously considered to take place in cytoplasm, as mature miRNAs were observed to be exported to the cytoplasm for Argonaute (AGO) loading and subsequent target binding. Recently, this notion was challenged by increasing pieces of evidence in the animal cells that uncovered the nuclear importation and action of the AGO-associated miRNAs. The nuclear-localized regulatory mode was also reported for the plant miRNAs. However, evidence is still lacking to show the universality and conservation of the miRNA-mediated regulation in the plant nuclei. Here, we introduced a bioinformatics workflow for genome-wide investigation of miRNA-guided, cleavage-based regulation of the nascent nuclear transcripts. Facilitated by the tool package PmiRNTSA (Plant microRNA-mediated nascent transcript slicing analyzer), plant biologists could perform a comprehensive search for the miRNA slicing sites located within the introns or the exon-intron/intron-exon junctions of the target transcripts, which are supported by degradome sequencing data. The results enable the researchers to examine the co-transcriptional regulatory model of the miRNAs for a specific plant species. Moreover, a case study was performed to search for the slicing sites located within the exon-intron/intron-exon junctions in two model plants. A case study was performed to show the feasibility and reliability of our workflow. Together, we hope that this work could inspire much more innovative research efforts to expand the current understanding of the miRNA action modes in plants.

Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Na+ exclusion in Arabidopsis thaliana.

Soil salinity is a common environmental stress factor that limits agricultural production worldwide. Plants have evolved different strategies to achieve salt tolerance. miR393 has been identified as closely related to biotic and abiotic stresses, and targets F-box genes that encode auxin receptors. The miR393-TIR1/AFB2/AFB3 regulatory module was discovered to have multiple functions that manipulate the auxin response. This study focused on miR393 and one of its targets, TIR1, and found that they played potential roles in response to salt stress. Our results showed that overexpression of a miR393-resistant TIR1 gene (mTIR1) in Arabidopsis clearly enhanced salt stress tolerance, which led to a higher germination rate, less water loss, reduced inhibition of root elongation, delayed senescence, decreased death rate and stabilized Chl content. These plants accumulated more proline and anthocyanin, and displayed enhanced osmotic stress tolerance. The expression of some salt stress-related genes was altered, and sodium content can be reduced in these plants under salt stress. We proposed that highly increased auxin signaling by overexpression of mTIR1 may trigger auxin-mediated downstream pathways to enhance plant salt stress resistance by osmoregulation and increased Na(+) exclusion.

Coordinated Transcriptome and Metabolome Analyses of a Barley hvhggt Mutant Reveal a Critical Role of Tocotrienols in Endosperm Starch Accumulation.

Tocotrienols and tocopherols (vitamin E) are potent antioxidants that are synthesized in green plants. Unlike ubiquitous tocopherols, tocotrienols predominantly accumulate in the endosperm of monocot grains, catalyzed by homogentiate geranylgeranyl transferase (HGGT). Previously, we generated a tocotrienol-deficient hvhggt mutant with shrunken barley grains. However, the relationship between tocotrienols and grain development remains unclear. Here, we found that the hvhggt lines displayed hollow endosperms with defective transfer cells and reduced aleurone layers. The carbohydrate and starch contents of the hvhggt endosperm decreased by approximately 20 and 23%, respectively. Weighted gene coexpression network analyses identified a critical gene module containing HvHGGT, which was strongly associated with the hvhggt mutation and enriched with gene functions in starch and sucrose metabolism. Metabolome measurements revealed an elevated soluble sugar content in the hvhggt endosperm, which was significantly associated with the identified gene modules. The hvhggt endosperm had significantly higher NAD(H) and NADP(H) contents and lower levels of ADPGlc (regulated by redox balance) than the wild-type, consistent with the absence of tocotrienols. Interestingly, exogenous α-tocotrienol spraying on developing hvhggt spikes partially rescued starch accumulation and endosperm defects. Our study supports a potential novel function of tocotrienols in grain starch accumulation and endosperm development in monocot crops.

Zearalenone regulates microRNA156 to affect the root development of Tetrastigma hemsleyanum.

Zearalenone (ZEN) is a secondary metabolite from Fusarium species. It is also present in plants and regulates the photochemical reaction in Photosystem II, the stress response and root growth. To investigate the mechanism by which ZEN regulates Tetrastigma hemsleyanum root growth, differentially expressed microRNAs (miRNAs) were identified and verified by high-throughput sequencing and Agrobacterium rhizogenes-mediated transformation of the roots of T. hemsleyanum seedlings treated with and without ZEN. The predicted functions of microRNA156b (miR156b) and microRNA156f (miR156f) were confirmed in transgenic hairy roots. (i) A total of 70 miRNAs showed significantly different expression levels under ZEN treatment, including seven highly conserved miRNAs. (ii) The number of lateral roots and total root length of the transgenic hairy roots overexpressing miR156b and miR156f was significantly higher than the wild-type hairy roots, and thus the overexpression of miR156b and miR156f in T. hemsleyanum promoted lateral root development. (iii) Bioinformatics analysis predicted that the target genes of miR156b and miR156f were SPL9/10. As compared with the wild-type hairy roots, the expression of SPL9 was significantly lower in the hairy roots overexpressing miR156b, and the expression of SPL10 was significantly lower in the hairy roots overexpressing miR156f. Therefore, SPL9 could be the target gene of miR156b, and SPL10 could be the target gene of miR156f. This study shows that ZEN could increase the expression of miR156b and miR156f in T. hemsleyanum roots, which negatively regulated the expression of their putative target genes SPL9 and SPL10, consequently promoting the growth and development of the lateral roots.

Deciphering the potential of a plant growth promoting endophyte Rhizobium sp. WYJ-E13, and functional annotation of the genes involved in the metabolic pathway.

Plant growth-promoting rhizobacteria (PGPR) are well-acknowledged root endophytic bacteria used for plant growth promotion. However, which metabolites produced by PGPR could promote plant growth remains unclear. Additionally, which genes are responsible for plant growth-promoting traits is also not elucidated. Thus, as comprehensive understanding of the mechanism of endophyte in growth promotion is limited, this study aimed to determine the metabolites and genes involved in plant growth-promotion. We isolated an endophytic Rhizobium sp. WYJ-E13 strain from the roots of Curcuma wenyujin Y.H. Chen et C. Ling, a perennial herb and medicinal plant. The tissue culture experiment showed its plant growth-promoting ability. The bacterium colonization in the root was confirmed by scanning electron microscopy and paraffin sectioning. Furthermore, it was noted that the WYJ-E13 strain produced cytokinin, anthranilic acid, and L-phenylalanine by metabolome analysis. Whole-genome analysis of the strain showed that it consists of a circular chromosome of 4,350,227 bp with an overall GC content of 60.34%, of a 2,149,667 bp plasmid1 with 59.86% GC, and of a 406,180 bp plasmid2 with 58.05% GC. Genome annotation identified 4,349 putative protein-coding genes, 51 tRNAs, and 9 rRNAs. The CDSs number allocated to the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and Clusters of Orthologous Genes databases were 2027, 3,175 and 3,849, respectively. Comparative genome analysis displayed that Rhizobium sp. WYJ-E13 possesses the collinear region among three species: Rhizobium acidisoli FH23, Rhizobium gallicum R602 and Rhizobium phaseoli R650. We recognized a total set of genes that are possibly related to plant growth promotion, including genes involved in nitrogen metabolism (nifU, gltA, gltB, gltD, glnA, glnD), hormone production (trp ABCDEFS), sulfur metabolism (cysD, cysE, cysK, cysN), phosphate metabolism (pstA, pstC, phoB, phoH, phoU), and root colonization. Collectively, these findings revealed the roles of WYJ-E13 strain in plant growth-promotion. To the best of our knowledge, this was the first study using whole-genome sequencing for Rhizobium sp. WYJ-E13 associated with C. wenyujin. WYJ-E13 strain has a high potential to be used as Curcuma biofertilizer for sustainable agriculture.

Digital RNA-seq transcriptome plus tissue anatomy analyses reveal the developmental mechanism of the calabash-shaped root in Tetrastigma hemsleyanum.

Tetrastigma hemsleyanum Diels & Gilg ex Diels is a liana plant with promising medicinal and ornamental values. Its calabash-shaped roots (CRs) are served as a traditional Chinese herb. However, it takes a long growth period to form CRs. In this study, three types of architectural roots, including fibrous roots (FRs), bar-shaped roots (BRs) and CRs, were employed as materials, and the characteristics of histo-anatomy and digital RNA-seq transcriptome profiles were analyzed. Among the three types of roots, the vascular bundles in FRs were intact, while some of the vascular bundles degenerated in BRs, and only few traces of vascular bundles existed in CRs. Meanwhile, no obvious cell inclusions were found in the cytoplasm of FRs, while a few inclusions were found in BRs, and abundant inclusions were detected in CRs, which might be the main source of medicinal components in roots. The transcriptome profiles and qRT-PCR validation indicated that seven upregulated genes, encoding xyloglucan glycosyltransferase, ACC oxidase, CYP711A1, SHORT-ROOT transcript factor, galacturonosyltransferas, WAT1 and WRKY, and two downregulated genes, encoding LRR receptor-like serine/threonine-protein kinase and CYP83B1, were probably involved in the formation and development of CRs. In addition, Gene Ontology terms of intrinsic component of membrane, integral component of membrane, cell periphery, membrane part, plasma membrane, membrane, intrinsic component of plasma membrane, cellular chemical homeostasis and plasma membrane part were probably related to the formation of CRs. Kyoto Encyclopedia of Genes and Genomes pathways related to the development of CRs probably included MAPK signaling pathway-plant, plant hormone signal transduction and circadian rhythm-plant. Our finding suggested a probable mode for the formation of CRs.

[Construction of a novel vector harboring green fluorescence protein gene (gfp) and high expression of gfp in transformed roots of Petunia hybrida].

A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida. The high activity of green fluo-rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5' and 3' of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloning sites at 5' end and one-sites of EcoRand Bsmsites at 3' end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transforma-tion.

Sequence analysis of a novel insertion site of transposon IS10.

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5'-CTGAGAGATCCCCTCATAATTT-3' and 5'-AAATCATTAGGGGATTCATCAG-3', which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5'-TGCTTGGTT-3' instead of the previously reported 5'-NGCTNAGCN-3'. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5alpha and that there were two copies in the DH5alpha genome.

[Hairy root induced by wild-type agrobacterium rhizogenes K599 in soybean, cucumber and garden balsam in vivo].

The plantlets of soybean, cucumber and garden balsam were inoculated by wild-type Agrobacterium rhizogenes K599, and hairy root was induced on inoculated sites in vivo. The frequencies of hairy root induction from wound cotyledons of soybean, cucumber and garden balsam were 100%, 65% and 91%, respectively. Moreover, hairy root was induced from healthy cucumber axillary bud with frequency of 10%. PCR analysis of hairy root DNA was conducted using the primers from rolC gene. The PCR results showed that all hairy root lines contained T-DNA. The established system should be ideal for studying soybean and cucumber nematode and garden balsam breeding of flower dwarf architecture.

[Cloning and sequencing of fragments associated with cytoplasm male sterility of rice].

Mitochorndrial DNA (mtDNA), nuclear DNA (nDNA), and genomic DNA (gDNA) were individually extracted from Zhenshan 97A, Xieqingzao A and A-23, which were cytoplasmic male sterile (wild abortive, dwarf abortive and BT), and from Zhenshan 97B, Xieqingzao B and BT, which were maintainers lines. Each of them was analyzed with 500 random primers by RAPD. Six fragments specific to male sterile lines were amplified. A fragment, PWH-17, associated with wild and dwarf abortive type cytoplasmic male sterility was analyzed by Southern blotting and sequencing, and also tested by SCAR. It was 1879 bp in length, which contained 6 open reading frames and 8 repeated sequences. BLAST search revealed that partial sequence in PWH-17 showed high similarity to the upstream sequence of tRNA-Asp gene in Elytrigia elongate (GenBank accession number U14335) and wheat (X13243). The similarity ratios were 97% and 100%. It was inferred that the possible position of PWH-17 in rice (wild abortive) could be located between tRNA-Asp and Cox II, which will be discussed further.

[A template DNA preparation method for PCR and RAPD reaction by using alkali-treated wheat leaf tissues].

In this paper we introduce a new method of PCR and RAPD reaction by using a small quantity of wheat leaf tissue with alkali treatment. It can be used either to detect target gene or to analyze polymorphism of wheat varieties. This method is simple and reliable, especially for a large-scale detection.

[Lyophilized mixture of PCR ingredients and its application].

In this paper we introduce a new storage method of PCR ingredient. PCR mixture except DNA template has been frozen to dry powder by the DNA-Plus system. This kind of powder was stored at room temperature or 4 degrees. PCR has been run in different period of storage. It was discovered that the samples of lyophilization could keep activity for a long time.

[Transgenic plant regeneration of tobacco (Nicotiana tabacum) haboring mammalian cyp2e1 gene].

CYP2E1 enzyme encoded by cyp2e1 gene plays an important role in metabolism of heterogeneous organics in mammalian liver cells. The transgenic plant with cyp2e1 can metabolize various low molecular weight organic pollutants. However, it is unclear the mechanism of expression control of cyp2e1 in transgenic plant. In this study, plasmid pSLD50-6 with cyp2e1 and pKH200 with gus as control were transformed into Agrobacterium tumefaciens GV3101 separately. Then, the cyp2e1 or gus genes were transferred into tobacco (Nicotiana tabacum) and the transgenic plants were regenerated via Agrobacterium tumefaciens method. Real-time quantitative PCR (qRT-PCR) was used to analyze the cyp2e1 gene expression. The expression of cyp2e1 in transgenic tobacco with cyp2e1 decreased obviously treated by ethyl alcohol and reduced slightly by benzene and toluene, while it enhanced by acetone, formaldehyde and oxygen deficit in different levels. In addition, the gene expression of NADPH-P450 oxidoreductase and cytochrome b5 enzyme in the transgenic tobacco with cyp2e1 were increased significantly treated by benzene, which showed that NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic tobacco have relation with CYP2E1 detoxication process. It suggested that the NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic plant formed the requirement in mammalian and participated in the electron transport chain of CYP2E1 enzyme catalytic process.

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde.

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.