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  • 1. Arnqvist, L
    et al.
    Dutta, P C
    Jonsson, Lisbeth
    Södertörns högskola, Avdelning Naturvetenskap.
    Sitbon, F
    Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA2003Ingår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 131, nr 4, s. 1792-1799Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transgenic potato (Solanum tuberosum cv Desiree) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.

  • 2. Arnqvist, Lisa
    et al.
    Persson, Mattias
    Jonsson, Lisbeth
    Södertörns högskola, Institutionen för livsvetenskaper.
    Dutta, Paresh C.
    Sitbon, Folke
    Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol2008Ingår i: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 227, nr 2, s. 309-317Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008-1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008-1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.

  • 3.
    Delp, Gabriele
    et al.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Gradin, Therese
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Ahman, Inger
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Microarray analysis of the interaction between the aphid Rhopalosiphum padi and host plants reveals both differences and similarities between susceptible and partially resistant barley lines2009Ingår i: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 281, nr 3, s. 233-248Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins.

  • 4. Glinwood, R.
    et al.
    Gradin, Therese
    Karpinska, Barbara
    Södertörns högskola, Institutionen för livsvetenskaper.
    Ahmed, E.
    Jonsson, Lisbeth
    Södertörns högskola, Institutionen för livsvetenskaper.
    Ninkovic, V.
    Aphid acceptance of barley exposed to volatile phytochemicals differs between plants exposed in daylight and darkness2007Ingår i: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 2, nr 5, s. 321-326Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is well known that volatile cues from damaged plants may induce resistance in neighboring plants. Much less is known about the effects of volatile interaction between undamaged plants. In this study, barley plants, Hordeum vulgare cv. Kara, were exposed to volatiles from undamaged plants of barley cv. Alva or thistle Cirsium vulgare, and to the volatile phytochemicals, methyl salicylate or methyl jasmonate. Exposures were made either during natural daylight or darkness. Acceptance of exposed plants by the aphid Rhopalosiphum padi was assessed, as well as the expression of putative marker genes for the different treatments. Aphid acceptance of plants exposed to either barley or C. vulgare was significantly reduced, and an effect of the volatiles from undamaged plants was confirmed by the induction of pathogenesis-related protein, PR1a in exposed plants. However the effect on aphid acceptance was seen only when plants were exposed during darkness, whereas PR1a was induced only after treatment during daylight. Aphid acceptance of plants exposed to either methyl salicylate or methyl jasmonate was significantly reduced, but only when plants were exposed to the chemicals during daylight. AOS2 (allene oxide synthase) was induced by methyl jasmonate and BCI-4 (barley chemical inducible gene-4) by methyl salicylate in both daylight and darkness. It is concluded that (a) the effects on aphids of exposing barley to volatile phytochemicals was influenced by the presence or absence of light and (b) the response of barley to methyl salicylate/methyl jasmonate and to volatiles from undamaged plants differed at the gene and herbivore level.

  • 5.
    Larsson, Kristina A E
    et al.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Saheed, S A
    Gradin, Therese
    Södertörns högskola, Institutionen för livsvetenskaper.
    Delp, Gabriele
    Södertörns högskola, Institutionen för livsvetenskaper.
    Botha, C E J
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Tissue localization and aphid-induced regulation of N-methyltransferase involved in the synthesis of gramine in barlyManuskript (preprint) (Övrigt vetenskapligt)
  • 6.
    Larsson, Kristina A. E.
    et al.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Saheed, Sefiu A.
    Gradin, Therese
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Delp, Gabriele
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Karpinska, Barbara
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Botha, Christiaan E. J.
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Differential regulation of 3-aminomethylindole/N-methyl-3-aminomethylindole N-methyltransferase and gramine in barley by both biotic and abiotic stress conditions2011Ingår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 49, nr 1, s. 96-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The expression of NMT (3-aminomethylindole/N-methyl-3-aminomethylindole N-methyltransferase; EC 2.1.1.), involved in the biosynthesis of the indole alkaloid gramine, was investigated in aphid-infested barley (Hordeum vulgare L). NMT is induced by methyl jasmonate and it was hypothesized that the gene would be more strongly upregulated in aphid-resistant barley. We examined the effects of feeding by three aphid species; Russian wheat aphid (Diuraphis noxia Mordvilko), rose-grain aphid (Metopolophium dirhodum Walker) and bird cherry-oat aphid (Rhopalosiphum padi L.) on barley genotypes with varying resistance characteristics. The barley genotypes selected included the cultivar Libra, known to upregulate gramine after feeding by Schizaphis graminum. Infestation by R. padi and M. dirhodum resulted in higher NMT expression in the doubled haploid line 5172-28:4 (DH28:4), which has moderate resistance against R. padi, but not in other aphid barley combinations. None of the aphid plant combinations had however increased gramine, suggesting that aphid-induction of gramine is specific to S. graminum. The increased abundance of NMT transcript in aphid-infested DH28:4 did not lead to higher amounts of NMT protein or NMT enzyme activity, neither did 200 times upregulation of NMT transcript in cotyledons incubated with methyl jasmonate, illustrating that even large differences measured at transcript level may have no metabolic consequences. Drought stress or treatments with abscisic acid did lead to higher gramine concentrations in several barley cultivars, but without any concomitant increase of NMT transcripts. Thus, the regulation of the biosynthetic pathway to gramine at transcript and metabolite level diverges during two different stress conditions.

  • 7.
    Larsson, Kristina A. E.
    et al.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Zetterlund, Ingvor
    Södertörns högskola, Institutionen för livsvetenskaper.
    Delp, Gabriele
    Södertörns högskola, Institutionen för livsvetenskaper.
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper.
    N-Methyltransferase involved in gramine biosynthesis in barley: Cloning and characterization2006Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 67, nr 18, s. 2002-2008Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The indole alkaloid gramine occurs in leaves of certain barley (Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase (MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 mu M and 184 mu M, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and NIT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the NIT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.

  • 8. Nikus, J
    et al.
    Esen, A
    Jonsson, Lisbeth M V
    Södertörns högskola, Avdelning Naturvetenskap.
    Cloning of a plastidic rye (Secale cereale) beta-glucosidase cDNA and its expression in Escherichia coli2003Ingår i: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 118, nr 3, s. 337-345Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cDNA for a beta-glucosidase (EC3.2.1.21) was isolated from rye (Secale cereale, cv Motto) and the sequence corresponding to the mature protein cloned into pET21a expression vector and used for transformation of Escherichia coli. The recombinant beta-glucosidase expressed in E. coli was recognized by antibodies to maize beta-glucosidase and exhibited the same kinetic properties on the endogenous substrates hydroxamic acid glucosides and artificial substrates as the native enzyme purified from rye. The enzyme monomer had an apparent molecular weight of about 67 kDa. The isolated cDNA was analysed with web-based chloroplast targeting prediction programs. The programs predicted a chloroplast targeting peptide with a cleavage site between amino acid 49 and 50. Sequence alignment of the plastidic rye beta-glucosidase showed that the putative sites for substrate specificity of maize Glu1, W378 and F198 (F197) are conserved in the rye enzyme, whereas F205, F466 and A467 of maize Glu1 are exchanged for histidine, glycine and serine, respectively, in rye. The plastidic beta-glucosidase is expressed in all plant parts and the highest levels were found in the coleoptile and mesocotyl.

  • 9.
    Saheed, S. A.
    et al.
    Rhodes University, Grahamstown, South Africa.
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Botha, C. E. J.
    Rhodes University, Grahamstown, South Africa.
    Symptomatic defence mechanisms include wound callose deposition after Rhopalosiphum padi L. infestation of barley leaves2008Ingår i: South African Journal of Botany, ISSN 0254-6299, E-ISSN 1727-9321, Vol. 74, nr 2, s. 378-378Artikel i tidskrift (Refereegranskat)
  • 10.
    Saheed, S. A.
    et al.
    Rhodes University, Grahamstown, South Africa.
    Larsson, Kristina A. E.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Delp, Gabriele
    Södertörns högskola, Institutionen för livsvetenskaper.
    Botha, C. E. J.
    Rhodes University, Grahamstown, South Africa.
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper.
    Bradley, G.
    Rhodes University, Grahamstown, South Africa.
    Wound callose synthesis in response to Russian wheat aphid and Bird cherry-oat aphid feeding on barley ev Clipper2007Ingår i: South African Journal of Botany, ISSN 0254-6299, E-ISSN 1727-9321, Vol. 73, nr 2, s. 310-310Artikel i tidskrift (Refereegranskat)
  • 11. Saheed, S. A.
    et al.
    Liu, L.
    Jonsson, Lisbeth
    Södertörns högskola, Institutionen för livsvetenskaper.
    Botha, C. E. J.
    Xylem - as well as phloem sustains severe damage due to feeding by the Russian wheat aphid2007Ingår i: South African Journal of Botany, ISSN 0254-6299, E-ISSN 1727-9321, Vol. 73, nr 4, s. 593-599Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Investigation of comparative effects of feeding damage by the Russian wheat aphid (RWA, biotype SAI, Diuraphis noxia Mordvilko) on leaf blades of susceptible and resistant wheat cultivars (Triticum aestivum L. var Betta and Betta-Dn1 respectively) were carried out to establish the level of ultrastructural damage caused by this aphid and the possible limitation of damage induced which could be ascribed to the resistance gene Dn1 over the susceptible cultivar. Ultrastructurally, Betta-Dn1 sustained less damage to the vascular tissue as well as to the mesophyll during the experimental period. Both inter- and intracellular probes resulted in considerable saliva deposition as the aphids probed for suitable feeding sites. Salivary tracks were observed between and within mesophyll, bundle sheath cells as well as the vascular tissue, including the xylem. Disruption of organelles and cytoplasm resulted from cell probing and sheath deposition. Cell and organelle damage was more evident in the non-resistant Betta cultivar. The aphids probed for and fed from thin-walled sieve tubes preferentially. Few thick-walled sieve tubes showed evidence of either aphid probing or feeding-related damage. Saliva was deposited when the aphids probed inter- and intracellularly for feeding sites. The aphids appeared preferentially to probe for and feed from thin-walled sieve tubes, as few thick-walled sieve tubes showed evidence of damage. Vessels, apparently probed for water, contained watery saliva that encased the secondary walls and scaled pit membranes between probed vessels and xylem parenchyma. The xylem probed by the RWA was rendered non-functional, probably contributing to symptoms of leaf roll, chlorosis and necrosis, which were observed within two weeks of infestation in the susceptible Betta cultivar. This damage was limited in the resistant Betta-Dn] cultivar during the same time frame.

  • 12. Saheed, Sefiu A.
    et al.
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Botha, Christaan E. J.
    Russian wheat aphid causes greater reduction in phloem transport apacity of barley leaves than bird cherry-oat aphid.2010Ingår i: Acta Botanica Croatica, ISSN 0365-0588, E-ISSN 1847-8476, Vol. 69, nr 1, s. 7-18Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of feeding by the Russian wheat aphid (RWA). Dutraplu.s nom! ordvilko and the Bird cherry-oat aphid (BC A). Rhopaloppluan pad, L on he transport capacity of barley Holdeum vulgare L leaves were nvestigated and compated with a view to i-elan ng these effects to the isible symptoms shown by the respective infested plants RWA causes xtensive chlorosis and neciosis on an infested plant whereas I3CA auses no obseivable symptoms Our results using the xenobiotic. phloem obile Bum ophole. 5, 6 carboxyBurn escei n chacetate (5. 6-CFDA) evealed striking ch fletences in damage to the transpol t of ssimilates thiough the phloem by these two aphids The result clearly uggests that short-term feeding by RWA causes a reduction in tiansport f assimikites and a mole severe reduction oi pei haps even permanent essation of transport during long-term feed111,2. In contrast. feechntz y BCA does not lead to a !milked dect ease in transport do ring hort-term feeding period. howevei, a !eduction in the uanspoit was ecorded donne long-term feeding activities These iesults perhaps uggest that damage to ti ansport capacities of the barley leaves ppeals lobe partly responsible for the observed symptoms in WIA-infested plants and the lack of them during BCA in symptoms such as eduction ol cessation in transport of assionlates to growing tissues ay lead to such observable symptoms

  • 13. Saheed, Sefiu Adekilekun
    et al.
    Botha, Christiaan Edward Johannes
    Liu, Lin
    Jonsson, Lisbeth
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Comparison of structural damage caused by Russian wheat aphid (Diuraphis noxia) and Bird cherry-oat aphid (Rhopalosiphum padi) in a susceptible barley cultivar, Hordeum vulgare cv. Clipper2007Ingår i: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 129, nr 2, s. 429-435Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Russian wheat aphid (RWA, (Diuraphis noxia) and the Bird cherry-oat aphid (BCA, (Rhopalosiphum padi L.) cause severe damage to grain crops, including barley. An investigation of the effects of these aphids on a susceptible cultivar revealed that BCA-infested barley plants remained healthy looking for 2 weeks after feeding commenced. In contrast, signs of stress and damage, including chlorosis and leaf necrosis were evident in RWA-infested plants. Our study suggests that damage to the vascular tissue because of sustained feeding by BCA was not as extensive as that caused by RWA. In addition, there is a marked difference in the salivary secretion pattern within xylem elements punctured by aphids tapping the xylem for water. RWA deposit electron-dense, amorphous to smooth saliva, which completely encases the inner walls of affected elements, and saliva encases pit membranes between xylem elements, and between xylem vessels and xylem parenchyma. Xylem tapped by BCA contained more granular saliva, which apparently does not occlude vessel wall apertures or the pit membranes to the same extent, as was observed with RWA. Damage to phloem tissue, including phloem parenchyma elements, sieve tube-companion cell (CC-ST) complexes as well as thick-walled ST, was extensive. Plasmodesmata between phloem parenchyma elements as well as pore plasmodesmata between the CC and ST were occluded by callose. We conclude that severe, perhaps permanent damage to conducting elements in RWA-infested leaves may be responsible for the detrimental chlorosis and necrosis symptoms. These symptoms are absent in BCA-infested plants.

  • 14. Saheed, Sefiu
    et al.
    Cierlik, Izabela
    Södertörns högskola, Institutionen för livsvetenskaper.
    Larsson, Kristina A. E.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Delp, Gabriele
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Bradley, Graeme
    Jonsson, Lisbeth M. V.
    Södertörns högskola, Institutionen för livsvetenskaper, Biologi. Södertörns högskola, Institutionen för livsvetenskaper, Molekylärbiologi.
    Botha, Christiaan E. J.
    Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or beta-1,3-glucanase transcript abundance2009Ingår i: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 135, nr 2, s. 150-161Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of infestation by the bird cherry-oat aphid (BCA), (Rhopalosiphum padi L) and the Russian wheat aphid (RWA) (Diuraphis noxia Mordvilko) on callose deposition and transcription of genes related to callose accumulation were investigated in barley (Hordeum vulgare L. cv. Clipper). The BCA, which gives no visible symptoms, induced very limited callose deposition, even after 14 days of infestation. In contrast, RWA, which causes chlorosis, white and yellow streaking and leaf rolling, induced callose accumulation already after 24 h in longitudinal leaf veins. The deposition was pronounced after 72 h, progressing during 7 and 14 days of infestation. In RWA-infested source leaves, callose was also induced in longitudinal veins basipetal to the aphid-infested tissue, whereas in sink leaves, more callose deposition was found above the feeding sites. Eight putative callose synthase genes were identified in a database search, of which seven were expressed in the leaves, but with similar transcript accumulation in control and aphid-infested tissue. Five out of 12 examined beta-1,3-glucanases were expressed in the leaves. All five were upregulated in RWA-infested tissue, but only two in BCA-infested tissue, and to a lesser extent than by RWA. The results suggest that callose accumulation may be partly responsible for the symptoms resulting from RWA infestation and that a callose-inducing signal may be transported in the phloem. Furthermore, it is concluded that the absence of callose deposition in BCA-infested leaves is not because of a stronger upregulation of callose-degrading beta-1,3-glucanases in this tissue, as compared to RWA-infested leaves.

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