Nat Commun , IF:12.121 , 2019 Dec , V10 (1) : P5516 doi: 10.1038/s41467-019-13543-1
Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter.
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190, Wien, Austria.; Institute of Experimental Botany of the Czech Academy of Sciences, Rozvojova 263 Praha 6, 165 02, Praha, Czech Republic.; Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400, Klosterneuburg, Austria.; Boehringer Ingelheim RCV, Doktor-Boehringer-Gasse 5-11, 1120, Wien, Austria.; Department of Experimental Plant Biology, Faculty of Science, Charles University, 128 44, Prague 2, Prague, Czech Republic.; Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190, Wien, Austria. christian.luschnig@boku.ac.at.
Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.
PMID: 31797871
Environ Pollut , IF:6.792 , 2019 Dec , V255 (Pt 2) : P113256 doi: 10.1016/j.envpol.2019.113256
Glutaredoxin S25 and its interacting TGACG motif-binding factor TGA2 mediate brassinosteroid-induced chlorothalonil metabolism in tomato plants.
Department of Horticulture, Zijingang Campus, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, PR China.; College of Forestry, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang, PR China.; Department of Horticulture, Zijingang Campus, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, PR China; Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, 866 Yuhangtang Road, Hangzhou, 310058, PR China.; Institute of Pesticide & Environmental Toxicology, Zijingang Campus, Zhejiang University, Hangzhou, 310058, China.; Department of Horticulture, Zijingang Campus, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, PR China. Electronic address: xiaojianxia@zju.edu.cn.
The presence of pesticide residues in fresh fruits and vegetables poses a serious threat to human health. Brassinosteroids (BRs) can reduce pesticide residues in plants, but the underlying mechanisms still remain unclear. Here, we identified a tomato glutaredoxin gene GRXS25 which was induced by 24-epibrassinolide (EBR) and chlorothalonil (CHT) in a way dependent on apoplastic reactive oxygen species (ROS). Silencing of GRXS25 in tomato abolished EBR-induced glutathione S-transferases (GSTs) gene expression and activity, leading to an increased CHT residue. Yeast two-hybrid and bimolecular fluorescence complementation assays showed protein-protein interaction between GRXS25 and a transcription factor TGA2. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that TGA2 factor bound to the TGACG-motif in the GST3 promoter. While silencing of TGA2 strongly compromised, overexpression of TGA2 enhanced expression of GST genes and CHT residue metabolism. Our results suggest that BR-induced apoplastic ROS trigger metabolism of pesticide residue in tomato plants through activating TGA2 factor via GRXS25-dependent posttranslational redox modification. Activation of plant detoxification through physiological approaches has potential implication in improving the food safety of agricultural products.
PMID: 31563783
J Mater Chem B , IF:5.344 , 2019 Dec , V7 (48) : P7783-7794 doi: 10.1039/c9tb01985e
Polyphosphazene-based nanocarriers for the release of agrochemicals and potential anticancer drugs.
Institute of Polymer Chemistry (ICP), Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria. javenator@gmail.com.; Johannes Kepler University Linz, Kepler University Hospital GmbH, Department for Cardiac-, Vascular- and Thoracic Surgery, Altenberger Str. 69, 4040 Linz and Krankenhausstrasse 9, 4020 Linz, Austria.
The synthesis and characterisation of novel polyphosphazene nanocarriers, based on hydrophilic polyalkylene oxide Jeffamine M1000 and hydrophobic steroids with a glycinate linker for pH-controlled release of diosgenin and two brassinosteroids (DI31 and S7) with agrochemical and potential anticancer activity, is hereby described. Polyphosphazenes carrying approximately 17 wt% of DI31 or S7 self-assembled in water to form 120-150 nm nanoaggregates, which showed an excellent plant growth effect on radish cotyledons due to sustained delivery of approximately 30% of the agrochemicals after 4 days. Cytotoxic evaluation showed that all polymers carrying steroids and Jeffamine M1000 resulted in strong to moderate toxicity to MCF-7 cancer cells and were non-toxic to primary human lung fibroblast cells at 0.1 to 0.025 mg mL-1. Thus, DI31 and S7 bearing polymers applied at 10-4 to 10-6 mg mL-1 for delivery of recommended DI31 or S7 quantities to crops should be harmless to humans. Particularly, DI31 and S7 bearing polymers with strong cytotoxicity on MCF-7 and non-toxicity on primary human lung fibroblasts, good cell uptake after 6 hours, proper hydrodynamic sizes between 100 and 200 nm, and slow sustained release of cytotoxic drugs (DI31, S7) in acidic conditions might potentiate their accumulation in cancer tissues with good antitumour effects and minor side effects. These results demonstrated that preparation of brassinosteroid bearing polymers is a promising strategy for the preparation of better agrochemicals with reduced pollutant impact on sustainable agriculture and potential anticancer formulations based on analogues of brassinosteroids.
PMID: 31755890
Bioelectrochemistry , IF:4.722 , 2019 Dec , V130 : P107336 doi: 10.1016/j.bioelechem.2019.107336
Direct electrochemistry of bacterial surface displayed cytokinin oxidase and its application in the sensitive electrochemical detection of cytokinins.
Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, Key Lab for Biotechnology of National Commission for Nationalities, College of Life Science, South Central University for Nationalities, Wuhan 430074, PR China.; Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, Key Lab for Biotechnology of National Commission for Nationalities, College of Life Science, South Central University for Nationalities, Wuhan 430074, PR China. Electronic address: wuyunhua@mail.scuec.edu.cn.
Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E(0')) of -0.45V at pH7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0muM and a lower limit of detection of 0.7muM (S/N=3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.
PMID: 31377396
Int J Mol Sci , IF:4.556 , 2019 Dec , V21 (1) doi: 10.3390/ijms21010245
Cyclin-Dependent Kinase Inhibitors KRP1 and KRP2 Are Involved in Grain Filling and Seed Germination in Rice (Oryza sativa L.).
State Key Lab of Rice Biology, China National Rice Research Institute, Hangzhou 311400, China.; Biotechnology Unit, National Cereals Research Institute, Badeggi, Bida 912101, Nigeria.; College of Life Science, Yangtze University, Jingzhou 434025, China.
Cyclin-dependent kinase inhibitors known as KRPs (kip-related proteins) control the progression of plant cell cycles and modulate various plant developmental processes. However, the function of KRPs in rice remains largely unknown. In this study, two rice KRPs members, KRP1 and KRP2, were found to be predominantly expressed in developing seeds and were significantly induced by exogenous abscisic acid (ABA) and Brassinosteroid (BR) applications. Sub-cellular localization experiments showed that KRP1 was mainly localized in the nucleus of rice protoplasts. KRP1 overexpression transgenic lines (OxKRP1), krp2 single mutant (crkrp2), and krp1/krp2 double mutant (crkrp1/krp2) all exhibited significantly smaller seed width, seed length, and reduced grain weight, with impaired seed germination and retarded early seedling growth, suggesting that disturbing the normal steady state of KRP1 or KRP2 blocks seed development partly through inhibiting cell proliferation and enlargement during grain filling and seed germination. Furthermore, two cyclin-dependent protein kinases, CDKC;2 and CDKF;3, could interact with KRP1 in a yeast-two-hybrid system, indicating that KRP1 might regulate the mitosis cell cycle and endoreduplication through the two targets. In a word, this study shed novel insights into the regulatory roles of KRPs in rice seed maturation and germination.
PMID: 31905829
Int J Mol Sci , IF:4.556 , 2019 Dec , V20 (24) doi: 10.3390/ijms20246108
Proteome and Ubiquitome Changes during Rose Petal Senescence.
Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture, China Agricultural University, Beijing 100193, China.
Petal senescence involves numerous programmed changes in biological and biochemical processes. Ubiquitination plays a critical role in protein degradation, a hallmark of organ senescence. Therefore, we investigated changes in the proteome and ubiquitome of senescing rose (Rosa hybrida) petals to better understand their involvement in petal senescence. Of 3859 proteins quantified in senescing petals, 1198 were upregulated, and 726 were downregulated during senescence. We identified 2208 ubiquitinated sites, including 384 with increased ubiquitination in 298 proteins and 1035 with decreased ubiquitination in 674 proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that proteins related to peptidases in proteolysis and autophagy pathways were enriched in the proteome, suggesting that protein degradation and autophagy play important roles in petal senescence. In addition, many transporter proteins accumulated in senescing petals, and several transport processes were enriched in the ubiquitome, indicating that transport of substances is associated with petal senescence and regulated by ubiquitination. Moreover, several components of the brassinosteroid (BR) biosynthesis and signaling pathways were significantly altered at the protein and ubiquitination levels, implying that BR plays an important role in petal senescence. Our data provide a comprehensive view of rose petal senescence at the posttranslational level.
PMID: 31817087
J Agric Food Chem , IF:4.192 , 2019 Dec , V67 (49) : P13526-13533 doi: 10.1021/acs.jafc.9b05241
DcBAS1, a Carrot Brassinosteroid Catabolism Gene, Modulates Cellulose Synthesis.
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, College of Horticulture , Nanjing Agricultural University , Nanjing , Jiangsu 210095 , People's Republic of China.; Co-Innovation Center for Sustainable Forestry in Southern China, Bamboo Research Institute , Nanjing Forestry University , Nanjing , Jiangsu 210037 , People's Republic of China.
Brassinosteroids (BRs) are important phytohormones and play critical roles during the growth and development of the plant. Numerous studies on biosynthesis and the signaling pathway of BRs have been performed, while the report about the metabolism of BRs is limited to carrots. In this study, we identified a homologous gene of AtCYP734A1/BAS1 (DCAR_009214), named DcBAS1, from carrots based on the data of the genome. The Arabidopsis overexpression line hosting the DcBAS1 gene was a dwarf and had crinkled blades and shortened petioles. Exogenous BL treatment rescued its growth and stem elongation. In addition, overexpressing DcBAS1 inhibited the cellulose synthesis in transgenic Arabidopsis plants. Results of quantitative real-time polymerase chain reaction revealed that overexpression of DcBAS1 inhibited the expression levels of AtCESAs genes (AtCESA1, AtCESA3, and AtCESA6), which are involved in cellulose synthesis in primary cell walls. AtBES1, which can be active by BR signaling, was also inhibited. These results revealed that DcBAS1 is the important gene involved in BR metabolism in carrots. Overexpression of DcBAS1 reduced the level of endogenous BRs and inhibited the cellulose synthesis in transgenic Arabidopsis plants.
PMID: 31725271
Physiol Plant , IF:4.148 , 2019 Dec , V167 (4) : P526-539 doi: 10.1111/ppl.12969
Regulation of sterol content and biosynthetic gene expression during flower opening and early fruit development in olive.
Plant Physiology, Faculty of Science, University of Extremadura, Badajoz, 06006, Spain.; Instituto Nacional de Investigacao Agraria e Veterinaria, I.P., UEIS Biotecnologia e Recursos Geneticos, Elvas, 7351-901, Portugal.
Phytosterols are lipophilic membrane components essential not only for diverse cellular functions but also are biosynthetic precursors of the plant hormone, brassinosteroid (BR). However, the interaction between phytosterol and BR during early fleshy-fruit growth remains largely uncharacterized. In olive, phytosterols are important lipids because they affect oil quality, but phytosterol composition during flowering and early fruit development has not been explored. Here, we first investigated the temporal changes in phytosterol composition, and biosynthetic gene expression that occurred during olive flower opening and early fruit growth. Next, we analyzed the interrelationship between phytosterol and BR, whose levels we manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz). In this report, the profiling of phytosterol measurement revealed that beta-sitosterol is the most abundant in olive reproductive organs. Our data demonstrate that both OeCYP51 and OeSMT2 genes are upregulated during floral anthesis in good agreement with the rise in cholesterol and beta-sitosterol contents in olive flower. By contrast, the OeCYP51 and OeSMT2 genes displayed different expression patterns during early olive-fruit development. Furthermore, our data show that exogenous EBR enhanced the early olive-fruit growth, as well as the OeSMT2 transcript and beta-sitosterol levels, but decreased the OeCYP51 transcript, squalene, campesterol and cholesterol levels, whereas the Brz treatment exerted the opposite effect. Overall, our findings indicate an up-regulation of beta-sitosterol biosynthesis by BR at the transcriptional level during early olive-fruit growth, providing a valuable tool to unravel the physiological function of SMT2 in future studies.
PMID: 30912149
Planta , IF:3.39 , 2019 Dec , V251 (1) : P27 doi: 10.1007/s00425-019-03322-z
Mesocotyl elongation, an essential trait for dry-seeded rice (Oryza sativa L.): a review of physiological and genetic basis.
CAAS-IRRI Joint Laboratory for Genomics-Assisted Germplasm Enhancement, Agricultural Genomics Institute in Shenzhen, Chinese Academy of Agricultural Sciences (CAAS), Shenzhen, 518120, China.; Collaborative Innovation Center of Henan Grain Crops and Key Laboratory of Rice Biology in Henan Province, College of Agronomy, Henan Agricultural University, Zhengzhou, 450002, China.; CAAS-IRRI Joint Laboratory for Genomics-Assisted Germplasm Enhancement, Agricultural Genomics Institute in Shenzhen, Chinese Academy of Agricultural Sciences (CAAS), Shenzhen, 518120, China. hongyanliu@caas.cn.; Strategic Innovation Platform, International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines.
MAIN CONCLUSIONS: (1) Mesocotyl elongation is responsive to abiotic stresses, such as deep sowing drought, submergence, chilling, and salinity. (2) Humus soil culture with a burial depth of 6 cm and at the temperature of 30 degrees C could be the optimum method for mesocotyl length phenotyping, The frequently colocalized quantitative trait loci (QTL) controlling mesocotyl elongation were located on chromosome (3) 1 (RM562-RG146), chromosome 2 (RZ288-RM145), and chromosome 3 (RM426-RM520). Dry direct-seeding is becoming a popular rice cultivation technology in many countries, which reduces water use and labor costs enormously. Meanwhile, direct-seeding rice is also facing the problems of low seedling emergence rate, poor seedling establishment, weed infestation, and high crop lodging rate. To take the full advantages of direct-seeding, both agronomic and genetic solutions are needed. Varieties with optimum mesocotyl length are desired for improving rice seedling emergence rate, particularly under deep sowing and submergence, which is adopted to reduce lodging and increase tolerance to abiotic stresses. In this review, we summarized the physiological and genetic mechanisms of mesocotyl elongation in rice. The elongation of mesocotyl is affected by light, temperature, and water, and, as a result, is responsive to sowing depth, water content, and soil salinity. Plant hormones such as abscisic acid (ABA), brassinosteroid (BR), strigolactones (SLs), cytokinin (CTK), ethylene (ETH), jasmonic acid (JA), gibberellin (GA), and indole-3-acetic acid (IAA) play important roles in regulating mesocotyl elongation. A humus soil culture protocol developed by our team was shown to be a better high-throughput method for measuring mesocotyl length in large scale. Sixty-seven QTL controlling mesocotyl length were reported, which are distributed on all the 12 chromosomes. Twelve chromosomal regions were repeatedly found to have QTL using various mapping populations and methods. These regions should be targeted in future studies to isolate genes and develop markers for molecular breeding. Two genes with very different molecular functions have been cloned, highlighting the genetic complexity of mesocotyl elongation.
PMID: 31802259
Planta , IF:3.39 , 2019 Dec , V250 (6) : P1849-1865 doi: 10.1007/s00425-019-03268-2
Transcription factor OsNF-YB9 regulates reproductive growth and development in rice.
National Institute of Plant Genome Research, New Delhi, 110067, India.; Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, India.; National Institute of Plant Genome Research, New Delhi, 110067, India. pinky.agarwal@nipgr.ac.in.; National Institute of Plant Genome Research, New Delhi, 110067, India. akhilesh@genomeindia.org.; Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, India. akhilesh@genomeindia.org.
MAIN CONCLUSION: OsNF-YB9 controls heading by affecting expression of regulators of flowering. It affects the development of the reproductive meristem by interacting with MADS1 and controlling expression of hormone-related genes. Nuclear Factor-Y (NF-Y) family of transcription factors takes part in many aspects of growth and development in eukaryotes. They have been classified into three subunit classes, namely, NF-YA, NF-YB and NF-YC. In plants, this transcription factor family is much diverged and takes part in several developmental processes and stress. We investigated NF-Y subunit genes of rice (Oryza sativa) and found OsNF-YB9 as the closest homologue of LEAFY COTYLEDON1. OsNF-YB9 delayed the heading date when ectopically expressed in rice. Expression of several heading date regulating genes such as Hd1, Ehd1, Hd3a and RFT1 were altered. OsNF-YB9 overexpression also resulted in morphological defects in the reproductive organs and led to pseudovivipary. OsNF-YB9 interacted with MADS1, a key regulator of floral development. This NF-Y subunit acted upstream to several transcription factors as well as signalling proteins involved in brassinosteroid and gibberellic acid metabolism and cell cycle. OsNF-YB9 and OsNF-YC12 interacted in planta and the latter also delayed heading in rice upon overexpression suggesting its involvement in a similar pathway. Our data provide new insights into the rice heading date pathway integrating these OsNF-Y subunit members to the network. These features can be exploited to improve vegetative growth and yield of rice plants in future.
PMID: 31482329
Molecules , IF:3.267 , 2019 Dec , V24 (24) doi: 10.3390/molecules24244612
Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5alpha-Cholane Type Analogs.
Departamento de Quimica, Universidad Tecnica Federico Santa Maria, Avenida Espana 1680, Valparaiso 224000, Chile.; Instituto de Ciencias Quimicas Aplicadas, Facultad de Ingenieria, Universidad Autonoma de Chile, El Llano Subercaseaux 2801, Santiago 8900000, Chile.; Teodor Parella, Servei de Ressonancia Magnetica Nuclear, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Catalonia, Spain.
Natural brassinosteroids possess a 22R, 23R configuration that appears essential for biological activity. It is, therefore, interesting to elucidate if the activity of brassinosteroids with a short side chain depends on the C22 configuration. Herein, we describe the synthesis of new brassinosteroids analogs with 24-norcholane type of side chain and R configuration at C22. The initial reaction is the dihydroxylation of a terminal olefin that leads to S/R epimers. Three different methods were tested in order to evaluate the obtained S/R ratio and the reaction yields. The results indicate that Upjohn dihydroxylation is the most selective reaction giving a 1.0:0.24 S/R ratio, whereas a Sharpless reaction leads to a mixture of 1.0:0.90 S/R with 95% yield. Using the latter mixture and following a previous reported method, benzoylated derivatives and both S and R brassinosteroids analogs were synthesized. All synthesized compounds were completely characterized by NMR spectroscopy, and HRMS of new compounds are also given. In conclusion, a synthetic route for preparation of new analogs of brassinosteroids of 24-norcholane type and R configuration at C22 were described. It is expected that this will help to elucidate if a configuration at C22 is a structural requirement for hormonal growth activity in plants.
PMID: 31861056
Plants (Basel) , IF:2.762 , 2019 Dec , V8 (12) doi: 10.3390/plants8120614
Transcriptomic Analysis of Dark-Induced Senescence in Bermudagrass (Cynodon dactylon).
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.; CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China.; Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Wuhan 430074, China.; National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, College of Resources and Environment, Shandong Agricultural University, Daizong Road, Tai'an 271018, China.; College of Horticulture, Agricultural University of Hebei, Baoding 071001, China.
Leaf senescence induced by prolonged light deficiency is inevitable whenever turfgrass is cultivated in forests, and this negatively influences the survival and aesthetic quality of the turfgrass. However, the mechanism underlying dark-induced senescence in turfgrass remained obscure. In this study, RNA sequencing was performed to analyze how genes were regulated in response to dark-induced leaf senescence in bermudagrass. A total of 159,207 unigenes were obtained with a mean length of 948 bp. The differential expression analysis showed that a total of 59,062 genes, including 52,382 up-regulated genes and 6680 down-regulated genes were found to be differentially expressed between control leaves and senescent leaves induced by darkness. Subsequent bioinformatics analysis showed that these differentially expressed genes (DEGs) were mainly related to plant hormone (ethylene, abscisic acid, jasmonic acid, auxin, cytokinin, gibberellin, and brassinosteroid) signal transduction, N-glycan biosynthesis, and protein processing in the endoplasmic reticulum. In addition, transcription factors, such as WRKY, NAC, HSF, and bHLH families were also responsive to dark-induced leaf senescence in bermudagrass. Finally, qRT-PCR analysis of six randomly selected DEGs validated the accuracy of sequencing results. Taken together, our results provide basic information of how genes respond to darkness, and contribute to the understanding of comprehensive mechanisms of dark-induced leaf senescence in turfgrass.
PMID: 31861053
Plant Biotechnol (Tokyo) , IF:0.901 , 2019 Dec , V36 (4) : P253-263 doi: 10.5511/plantbiotechnology.19.1030a
Characterization of steroid 5alpha-reductase involved in alpha-tomatine biosynthesis in tomatoes.
Graduate School of Agricultural Science, Kobe University, 1-1 Rokkoudai, Nada, Kobe, Hyogo 657-8501, Japan.; Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.; RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.; Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.; Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
alpha-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of alpha-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5alpha reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3betaHSD is involved in the three reactions except for C5alpha reduction, and in the present study, we aimed to elucidate the gene responsible for the C5alpha reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5alphaR1 and SlS5alphaR2, which show high homology with DET2, a brassinosteroid 5alpha reductase of Arabidopsis thaliana. The expression pattern of SlS5alphaR2 is similar to those of SGA biosynthetic genes, while SlS5alphaR1 is ubiquitously expressed, suggesting the involvement of SlS5alphaR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5alphaR1 and SlS5alphaR2 catalyze the reduction of tomatid-4-en-3-one at C5alpha to yield tomatid-3-one. Then, SlS5alphaR1- or SlS5alphaR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5alphaR2-knockout hairy roots, the alpha-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of alpha-tomatine was observed in the SlS5alphaR1-knockout hairy root. These results indicate that SlS5alphaR2 is responsible for the C5alpha reduction in alpha-tomatine biosynthesis and that SlS5alphaR1 does not significantly contribute to alpha-tomatine biosynthesis.
PMID: 31983879