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Journal Cover Plant Molecular Biology
  [SJR: 1.915]   [H-I: 137]   [9 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 1573-5028 - ISSN (Online) 0167-4412
   Published by Springer-Verlag Homepage  [2335 journals]
  • The monomeric GTPase RabA2 is required for progression and maintenance of
           membrane integrity of infection threads during root nodule symbiosis
    • Authors: Virginia Dalla Via; Soledad Traubenik; Claudio Rivero; O. Mario Aguilar; María Eugenia Zanetti; Flavio Antonio Blanco
      Pages: 549 - 562
      Abstract: Key message Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth. Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-016-0581-5
      Issue No: Vol. 93, No. 6 (2017)
  • The mobile RNAs, StBEL11 and StBEL29 , suppress growth of tubers in potato
    • Authors: Tejashree H. Ghate; Pooja Sharma; Kirtikumar R. Kondhare; David J. Hannapel; Anjan K. Banerjee
      Pages: 563 - 578
      Abstract: Key message We demonstrate that RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5 in potato. Both these RNAs appear to inhibit tuber growth by repressing the activity of target genes of StBEL5 in potato. Moreover, upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in selective organs. There are thirteen functional BEL1-like genes in potato that encode for a family of transcription factors (TF) ubiquitous in the plant kingdom. These BEL1 TFs work in tandem with KNOTTED1-types to regulate the expression of numerous target genes involved in hormone metabolism and growth processes. One of the StBELs, StBEL5, functions as a long-distance mRNA signal that is transcribed in leaves and moves into roots and stolons to stimulate growth. The two most closely related StBELs to StBEL5 are StBEL11 and -29. Together these three genes make up more than 70% of all StBEL transcripts present throughout the potato plant. They share a number of common features, suggesting they may be co-functional in tuber development. Upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short-days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in specific organs. Using a transgenic approach and heterografting experiments, we show that both these StBELs inhibit growth in correlation with the long distance transport of their mRNAs from leaves to roots and stolons, whereas suppression lines of these two RNAs exhibited enhanced tuber yields. In summary, our results indicate that the RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5. Both these RNAs appear to inhibit growth in tubers by repressing the activity of target genes of StBEL5.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-016-0582-4
      Issue No: Vol. 93, No. 6 (2017)
  • Heme oxygenase 1 defects lead to reduced chlorophyll in Brassica napus
    • Authors: Lixia Zhu; Zonghui Yang; Xinhua Zeng; Jie Gao; Jie Liu; Bin Yi; Chaozhi Ma; Jinxiong Shen; Jinxing Tu; Tingdong Fu; Jing Wen
      Pages: 579 - 592
      Abstract: We previously described a Brassica napus chlorophyll-deficient mutant (ygl) with yellow-green seedling leaves and mapped the related gene, BnaC.YGL, to a 0.35 cM region. However, the molecular mechanisms involved in this chlorophyll defect are still unknown. In this study, the BnaC07.HO1 gene (equivalent to BnaC.YGL) was isolated by the candidate gene approach, and its function was confirmed by genetic complementation. Comparative sequencing analysis suggested that BnaC07.HO1 was lost in the mutant, while a long noncoding-RNA was inserted into the promoter of the homologous gene BnaA07.HO1. This insert was widely present in B. napus cultivars and down-regulated BnaA07.HO1 expression. BnaC07.HO1 was highly expressed in the seedling leaves and encoded heme oxygenase 1, which was localized in the chloroplast. Biochemical analysis showed that BnaC07.HO1 can catalyze heme conversion to form biliverdin IXα. RNA-seq analysis revealed that the loss of BnaC07.HO1 impaired tetrapyrrole metabolism, especially chlorophyll biosynthesis. According, the levels of chlorophyll intermediates were reduced in the ygl mutant. In addition, gene expression in multiple pathways was affected in ygl. These findings provide molecular evidences for the basis of the yellow-green leaf phenotype and further insights into the crucial role of HO1 in B. napus.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-017-0583-y
      Issue No: Vol. 93, No. 6 (2017)
  • Transcriptomic and hormone analyses reveal mechanisms underlying petal
           elongation in Chrysanthemum morifolium ‘Jinba’
    • Authors: Jingjing Wang; Haibin Wang; Lian Ding; Aiping Song; Feng Shen; Jiafu Jiang; Sumei Chen; Fadi Chen
      Pages: 593 - 606
      Abstract: Key message Auxin regulates chrysanthemum petal elongation by promoting cell elongation. Transcriptomic analysis shows that auxin signal transduction may connect with other transcription factors by TCPs to regulate chrysanthemum petal elongation. As an ornamental species, Chrysanthemum morifolium has high ornamental and economic value. Petal size is the primary factor that influences the ornamental value of chrysanthemum, but the mechanism underlying the development of C. morifolium petals remains unclear. In our study, we tracked the growth of petals and found that the basal region of ‘Jinba’ petals showed a higher elongation rate, exhibiting rapid cell elongation during petal growth. During petal elongation growth, auxin was demonstrated to promote cell elongation and an increase in cell numbers in the petal basal region. To further study the molecular mechanisms underlying petal growth, the RNA-seq (high-throughput cDNA sequencing) technique was employed. Four cDNA libraries were assembled from petals in the budding, bud breaking, early blooming and full blooming stages of ‘Jinba’ flower development. Analysis of differentially expressed genes (DEGs) showed that auxin was the most important regulator in controlling petal growth. The TEOSINTEBRANCHED 1, CYCLOIDEA and PCF transcription factor genes (TCPs), basic helix-loop-helix-encoding gene (bHLH), glutaredoxin-C (GRXC) and other zinc finger protein genes exhibited obvious up-regulation and might have significant effects on the growth of ‘Jinba’ petals. Given the interaction between these genes in Arabidopsis thaliana, we speculated that auxin signal transduction might exhibit a close relationship with transcription factors through TCPs. In summary, we present the first comprehensive transcriptomic and hormone analyses of C. morifolium petals. The results offer direction in identifying the mechanism underlying the development of chrysanthemum petals in the elongated phase and have great significance in improving the ornamental characteristics of C. morifolium via molecular breeding.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-017-0584-x
      Issue No: Vol. 93, No. 6 (2017)
  • Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange
           ( Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker
    • Authors: Lorena Noelia Sendín; Ingrid Georgina Orce; Rocío Liliana Gómez; Ramón Enrique; Carlos Froilán Grellet Bournonville; Aldo Sergio Noguera; Adrián Alberto Vojnov; María Rosa Marano; Atilio Pedro Castagnaro; María Paula Filippone
      Pages: 607 - 621
      Abstract: Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-017-0586-8
      Issue No: Vol. 93, No. 6 (2017)
  • Depletion of abscisic acid levels in roots of flooded Carrizo citrange (
           Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) plants is a
           stress-specific response associated to the differential expression of
           PYR/PYL/RCAR receptors
    • Authors: Vicent Arbona; Sara I. Zandalinas; Matías Manzi; Miguel González-Guzmán; Pedro L. Rodriguez; Aurelio Gómez-Cadenas
      Pages: 623 - 640
      Abstract: Soil flooding reduces root abscisic acid (ABA) levels in citrus, conversely to what happens under drought. Despite this reduction, microarray analyses suggested the existence of a residual ABA signaling in roots of flooded Carrizo citrange seedlings. The comparison of ABA metabolism and signaling in roots of flooded and water stressed plants of Carrizo citrange revealed that the hormone depletion was linked to the upregulation of CsAOG, involved in ABA glycosyl ester (ABAGE) synthesis, and to a moderate induction of catabolism (CsCYP707A, an ABA 8′-hydroxylase) and buildup of dehydrophaseic acid (DPA). Drought strongly induced both ABA biosynthesis and catabolism (CsNCED1, 9-cis-neoxanthin epoxycarotenoid dioxygenase 1, and CsCYP707A) rendering a significant hormone accumulation. In roots of flooded plants, restoration of control ABA levels after stress release was associated to the upregulation of CsBGLU18 (an ABA β-glycosidase) that cleaves ABAGE. Transcriptional profile of ABA receptor genes revealed a different induction in response to soil flooding (CsPYL5) or drought (CsPYL8). These two receptor genes along with CsPYL1 were cloned and expressed in a heterologous system. Recombinant CsPYL5 inhibited ΔNHAB1 activity in vitro at lower ABA concentrations than CsPYL8 or CsPYL1, suggesting its better performance under soil flooding conditions. Both stress conditions induced ABA-responsive genes CsABI5 and CsDREB2A similarly, suggesting the occurrence of ABA signaling in roots of flooded citrus seedlings. The impact of reduced ABA levels in flooded roots on CsPYL5 expression along with its higher hormone affinity reinforce the role of this ABA receptor under soil-flooding conditions and explain the expression of certain ABA-responsive genes.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-017-0587-7
      Issue No: Vol. 93, No. 6 (2017)
  • In Arabidopsis thaliana distinct alleles encoding mitochondrial RNA
           PROCESSING FACTOR 4 support the generation of additional 5′ termini of
           ccmB transcripts
    • Authors: Katrin Stoll; Christian Jonietz; Sarah Schleicher; Catherine Colas des Francs-Small; Ian Small; Stefan Binder
      Pages: 659 - 668
      Abstract: In plant mitochondria, the 5′ ends of many transcripts are generated post-transcriptionally. We show that the pentatricopeptide repeat (PPR) protein RNA PROCESSING FACTOR 4 (RPF4) supports the generation of extra 5′ ends of ccmB transcripts in Landsberg erecta (Ler) and a number of other Arabidopsis thaliana ecotypes. RPF4 was identified in Ler applying a forward genetic approach supported by complementation studies of ecotype Columbia (Col), which generates the Ler-type extra ccmB 5′ termini only after the introduction of the RPF4 allele from Ler. Studies with chimeric RPF4 proteins composed of various parts of the RPF4 proteins from Ler and Col identified differences in the N-terminal and central PPR motifs that explain ecotype-specific variations in ccmB processing. These results fit well with binding site predictions in ccmB transcripts based on the known determinants of nucleotide base recognition by PPR motifs.
      PubDate: 2017-04-01
      DOI: 10.1007/s11103-017-0591-y
      Issue No: Vol. 93, No. 6 (2017)
  • INDETERMINATE DOMAIN PROTEIN binding sequences in the 5′-untranslated
           region and promoter of the SCARECROW gene play crucial and distinct roles
           in regulating SCARECROW expression in roots and leaves
    • Authors: Atsushi Kobayashi; Satoshi Miura; Akiko Kozaki
      Abstract: SCARECROW (SCR) and SHORT-ROOT (SHR), which belong to the GRAS transcription factor family, are key regulators of root and leaf growth and development. Despite the importance of SCR expression for proper plant development, the mechanism of SCR regulation has not been clarified. A previous study showed that an INDETERMINATE DOMAIN transcription factor, JACKDAW (JKD), is essential for the expression of SCR in combination with SCR and SHR. In this study, we characterized possible binding sequences of INDETERMINATE DOMAIN PROTEIN in the 1.5 kb upstream region of SCR. Mutation in a binding sequence 340 bp upstream of the ATG increased transcriptional activation by JKD in transient assays using Arabidopsis cultured cells. However, the activity was not enhanced by SCR/SHR. Histochemical analysis of promoter activity in transgenic Arabidopsis plants carrying a fusion of the promoter and the β-glucronidase reporter gene showed that mutation of the −340 bp sequence eliminated most of the promoter activity, indicating that this sequence was indispensable for SCR expression. Promoter deletion of downstream sequences from −280 bp lost the enhanced activity by SCR/SHR in transient assays and activity in root tips and the bundle sheath (BS) in plants. Conversely, mutation at −480 bp did not significantly influence transcriptional activity in transient assays. However, most of SCR expression was lost except for the root tip in plants. The sequences around −1 kb appeared to regulate SCR expression negatively in plants. Together, these INDETERMINATE DOMAIN PROTEIN binding sequences have crucial and distinct functions in regulating SCR expression.
      PubDate: 2017-03-21
      DOI: 10.1007/s11103-016-0578-0
  • The small auxin-up RNA OsSAUR45 affects auxin synthesis and transport in
    • Authors: Yan-Xia Xu; Meng-Zhu Xiao; Yan Liu; Jun-Liang Fu; Yi He; De-An Jiang
      Abstract: Key message This research is the first to demonstrate that OsSAUR45 is involved in plant growth though affecting auxin synthesis and transport by repressing OsYUCCA and OsPIN gene expression in rice. Small auxin-up RNAs (SAURs) comprise a large multigene family and are rapidly activated as part of the primary auxin response in plants. However, little is known about the role of SAURs in plant growth and development, especially in monocots. Here, we report the biological function of OsSAUR45 in the model plant rice (Oryza sativa). OsSAUR45 is expressed in a tissue-specific pattern and is localized to the cytoplasm. Rice lines overexpressing OsSAUR45 displayed pleiotropic developmental defects including reduced plant height and primary root length, fewer adventitious roots, narrower leaves, and reduced seed setting. Auxin levels and transport were reduced in the OsSAUR45 overexpression lines, potentially because of decreased expression of Flavin-binding monooxygenase family proteins (OsYUCCAs) and PIN-FORMED family proteins (OsPINs). Exogenous auxin application rapidly induced OsSAUR45 expression and partially restored the phenotype of rice lines overexpressing OsSAUR45. These results demonstrate that OsSAUR45 is involved in plant growth by affecting auxin synthesis and transport through the repression of OsYUCCA and OsPIN gene expression in rice.
      PubDate: 2017-03-20
      DOI: 10.1007/s11103-017-0595-7
  • RNAi inhibition of feruloyl CoA 6′-hydroxylase reduces scopoletin
           biosynthesis and post-harvest physiological deterioration in cassava (
           Manihot esculenta Crantz) storage roots
    • Authors: Shi Liu; Ima M. Zainuddin; Herve Vanderschuren; James Doughty; John R. Beeching
      Abstract: Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24–72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava’s marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6′-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6′-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6′-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.
      PubDate: 2017-03-18
      DOI: 10.1007/s11103-017-0602-z
  • Identification of the ligand of Pru p 3, a peach LTP
    • Authors: Nuria Cubells-Baeza; Cristina Gómez-Casado; Leticia Tordesillas; Carmen Ramírez-Castillejo; María Garrido-Arandia; Pablo González-Melendi; María Herrero; Luis F. Pacios; Araceli Díaz-Perales
      Abstract: Key message Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.
      PubDate: 2017-03-15
      DOI: 10.1007/s11103-017-0590-z
  • The molecular mechanism underlying anthocyanin metabolism in apple using
           the MdMYB16 and MdbHLH33 genes
    • Authors: Haifeng Xu; Nan Wang; Jingxuan Liu; Changzhi Qu; Yicheng Wang; Shenghui Jiang; Ninglin Lu; Deyun Wang; Zongying Zhang; Xuesen Chen
      Abstract: Key message MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.
      PubDate: 2017-03-12
      DOI: 10.1007/s11103-017-0601-0
  • New insights into the operative network of FaEO, an enone oxidoreductase
           from Fragaria x ananassa Duch.
    • Authors: Gabriella Collu; Domenica Farci; Francesca Esposito; Francesca Pintus; Joanna Kirkpatrick; Dario Piano
      Abstract: The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.
      PubDate: 2017-03-11
      DOI: 10.1007/s11103-017-0597-5
  • A naturally occurring conditional albino mutant in rice caused by defects
           in the plastid-localized OsABCI8 transporter
    • Authors: Xiuyu Zeng; Ran Tang; Herong Guo; Shanwen Ke; Bin Teng; Yu-Hung Hung; Zhenjiang Xu; Xin-Ming Xie; Tzung-Fu Hsieh; Xiang-Qian Zhang
      Abstract: A wide range of molecules are transported across membranes by the ATP binding cassette (ABC) transporters. Plants possess a collection of ABC proteins bearing similarities to the components of prokaryotic multi subunit ABC transporters, designed as ABC group I. However the functions of most of them are not well understood. Here, we characterized a naturally occurring rice mutant that exhibited albino phenotype under continuous rainy days in the field, but gradually recovered to normal green after the rainy season. Molecular and genetic analyses revealed that the phenotypes were caused by a mutation in the OsABCI8 that encoded a member of the ABCI family. Subcellular localization demonstrated that OsABCI8 is a chloroplast ABC transporter. Expression of OsABCI8 is significantly enhanced in rainy days compared to sunny days. Besides defects in chloroplast development and chlorophyll biosynthesis, the mutant phenotype is accompanied by a higher accumulation of iron, suggesting that OsABCI8 is involved in iron transportation and/or homeostasis in rice. Our results demonstrate that OsABCI8 represents a conserved ABCI protein involved in transition metals transportation and/or homeostasis and suggest an important role of the plastid-localized OsABCI8 for chloroplast development.
      PubDate: 2017-03-11
      DOI: 10.1007/s11103-017-0598-4
  • Identification of alternatively spliced transcripts of rice phytochelatin
           synthase 2 gene OsPCS2 involved in mitigation of cadmium and arsenic
    • Authors: Natasha Das; Surajit Bhattacharya; Somnath Bhattacharyya; Mrinal K. Maiti
      Abstract: Key message The OsPCS2 exhibits root- and shoot-specific differential ratios of alternatively spliced transcripts in indica rice under Cd stress, and plays role in Cd and As stress tolerance and accumulation. Enzymatic activity of phytochelatin synthase (PCS) in plant produces phytochelatins, which help in sequestration of heavy metal(loid)s inside the cell vacuole to alleviate toxicity. Here we report that among the two PCS genes—OsPCS1 and OsPCS2 in indica rice (Oryza sativa) cultivar, the OsPCS2 produces an alternatively spliced OsPCS2b transcript that bears the unusual premature termination codon besides the canonically spliced OsPCS2a transcript. Root- and shoot-specific differential ratios of alternatively spliced OsPCS2a and OsPCS2b transcript expressions were observed under cadmium stress. Saccharomyces cerevisiae cells transformed with OsPCS2a exhibited increased cadmium (Cd) and arsenic (As) tolerance and accumulation, unlike the OsPCS2b transformed yeast cells. An intron-containing hairpin RNA-mediated gene silencing was carried out in endosperm-specific manner for efficient down-regulation of OsPCS genes in rice grains. Analysis of the transgenic rice lines grown under metal(loid) stress revealed almost complete absence of both OsPCS1 and OsPCS2 transcripts in the developing seeds coupled with the significant reduction in the content of Cd (~51%) and As (~35%) in grains compared with the non-transgenic plant. Taken together, the findings indicate towards a crucial role played by the tissue-specific alternative splicing and relative abundance of the OsPCS2 gene during heavy metal(loid) stress mitigation in rice plant.
      PubDate: 2017-03-11
      DOI: 10.1007/s11103-017-0600-1
  • NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis
           thaliana revisited through transgenic expression of engineered barley PORB
           mutant proteins
    • Authors: Frank Buhr; Abderrahim Lahroussi; Armin Springer; Sachin Rustgi; Diter von Wettstein; Christiane Reinbothe; Steffen Reinbothe
      Abstract: NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the ‘RED’ family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.
      PubDate: 2017-03-04
      DOI: 10.1007/s11103-017-0592-x
  • Cassava C-repeat binding factor 1 gene responds to low temperature and
           enhances cold tolerance when overexpressed in Arabidopsis and cassava
    • Authors: Dong An; Qiuxiang Ma; Hongxia Wang; Jun Yang; Wenzhi Zhou; Peng Zhang
      Abstract: Key message Cassava MeCBF1 is a typical CBF transcription factor mediating cold responses but its low expression in apical buds along with a retarded response cause inefficient upregulation of downstream cold-related genes, rendering cassava chilling-sensitive. Low temperature is a major abiotic stress factor affecting survival, productivity and geographic distribution of important crops worldwide. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important regulators of abiotic stress response in plants. In this study, MeCBF1, a CBF-like gene, was identified in the tropical root crop cassava (Manihot esculenta Crantz). The MeCBF1 encodes a protein that shares strong homology with DREB1As/CBFs from Arabidopsis as well as other species. The MeCBF1 was localized to the nucleus and is mainly expressed in stem and mature leaves, but not in apical buds or stem cambium. MeCBF1 expression was not only highly responsive to cold, but also significantly induced by salt, PEG and ABA treatment. Several stress-associated cis-elements were found in its promoter region, e.g., ABRE-related, MYC recognition sites, and MYB responsive element. Compared with AtCBF1, the MeCBF1 expression induced by cold in cassava was retarded and upregulated only after 4 h, which was also confirmed by its promoter activity. Overexpression of MeCBF1 in transgenic Arabidopsis and cassava plants conferred enhanced crytolerance. The CBF regulon was smaller and not entirely co-regulated with MeCBF1 expression in overexpressed cassava. The retarded MeCBF1 expression in response to cold and attenuated CBF-regulon might lead cassava to chilling sensitivity.
      PubDate: 2017-03-04
      DOI: 10.1007/s11103-017-0596-6
  • CRISPR/Cas9-mediated targeted mutagenesis in upland cotton ( Gossypium
           hirsutum L.)
    • Authors: Madhusudhana R. Janga; LeAnne M. Campbell; Keerti S. Rathore
      Abstract: The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas)9 protein system has emerged as a simple and efficient tool for genome editing in eukaryotic cells. It has been shown to be functional in several crop species, yet there are no reports on the application of this or any other genome editing technologies in the cotton plant. Cotton is an important crop that is grown mainly for its fiber, but its seed also serves as a useful source of edible oil and feed protein. Most of the commercially-grown cotton is tetraploid, thus making it much more difficult to target both sets of homeologous alleles. Therefore, in order to understand the efficacy of the CRISPR/Cas9 system to target a gene within the genome of cotton, we made use of a transgenic cotton line previously generated in our laboratory that had a single copy of the green fluorescent protein (GFP) gene integrated into its genome. We demonstrate, for the first time, the use of this powerful new tool in targeted knockout of a gene residing in the cotton genome. By following the loss of GFP fluorescence, we were able to observe the cells that had undergone targeted mutations as a result of CRISPR/Cas9 activity. In addition, we provide examples of the different types of indels obtained by Cas9-mediated cleavage of the GFP gene, guided by three independent sgRNAs. The results provide useful information that will help us target important native genes in the cotton plant in future.
      PubDate: 2017-03-03
      DOI: 10.1007/s11103-017-0599-3
  • Isolation and functional characterization of a methyl jasmonate-responsive
           3-carene synthase from Lavandula x intermedia
    • Authors: Ayelign M. Adal; Lukman S. Sarker; Ashley D. Lemke; Soheil S. Mahmoud
      Abstract: Key message A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni–NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17 µM and 2.01 s−1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k cat /K m = 0.56 µM−1s−1) than NPP (k cat /K m = 0.044 µM−1s−1). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8–72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.
      PubDate: 2017-03-03
      DOI: 10.1007/s11103-017-0588-6
  • Retraction note to: Brassica RNA binding protein ERD4 is involved in
           conferring salt, drought tolerance and enhancing plant growth in
    • Authors: Archana N. Rai; Srinath Tamirisa; K. V. Rao; Vinay Kumar; P. Suprasanna
      PubDate: 2017-01-18
      DOI: 10.1007/s11103-016-0574-4
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