Drought tolerance through overexpression of monoubiquitin in transgenic tobacco

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<ul><li><p>Journal of Plant Physiology 165 (2008) 17451755</p><p>Drought tolerance throughof monoubiquitin in transgenic tobacco</p><p>Qifang Guo1, Jin Zha</p><p>State Key Laboratory of Crop BiolTaian, Shandong, 271018, PR Chi</p><p>Received 31 March 2007; received</p><p>KEYWORDSDrought tolerance;</p><p>gel blot analysis, we found higher amounts of Ubprotein conjugates than in control(tobacco carrying a PBI GUS vector without Ta-Ub2) and wild-type (WT) lines.</p><p>be an effective strategy for enhancing drought tolerance.</p><p>production worldwide (Sio-Se Mardeh et al.,2006), which is also one of the most severeenvironmental stresses that affects almost all plantfunctions (Yamaguchi-Shinozaki et al., 2002). In-</p><p>ARTICLE IN PRESS</p><p>leaf stomatal conductance; Pn, net photosynthetic rate; Ub,ubiquitin; WT, wild-type tobacco.Corresponding author. Tel.: +86 538 8246166;</p><p>fax: +86 538 8242288.E-mail address: wangw@sdau.edu.cn (W. Wang).creasing evidence has indicated that the molecular</p><p>0176-1617/$ - see front matter &amp; 2007 Published by Elsevier GmbH.doi:10.1016/j.jplph.2007.10.002</p><p>1These authors contributed equally to this paper.&amp; 2007 Published by Elsevier GmbH.</p><p>Introduction</p><p>Drought is the primary limitation to wheatAbbreviations: E, transpiration rate; E1, Ub-activating en-</p><p>zyme; E2, Ub-conjugating enzyme; E3, Ubprotein ligase; Gs,However, free Ub levels did not signicantly differ in the 3 genotypes. Seeds fromtransgenic, Ub-overexpressing tobacco germinated faster and seedlings grew morevigorously than control and WT samples, both under drought and non-droughtconditions. Furthermore, CO2 assimilation of transgenic plants was signicantlyhigher under drought stress. Our results indicate that Ub may be involved in theresponse of plants to drought stress and that overexpression of monoubiquitin mightGene expression;Transgenic tobacco;Ubiquitin;Wheatng1, Qiang Gao, Shichao Xing, Feng Li, Wei Wang</p><p>ogy, College of Life Sciences, Shandong Agricultural University,na</p><p>in revised form 8 October 2007; accepted 9 October 2007</p><p>SummaryUbiquitin (Ub) is present in all eukaryotic species examined. It is a multifunctionalprotein and one of its main known functions is to tag proteins for selectivedegradation by the 26S proteasome. In this study, Ta-Ub2, a cDNA sequencecontaining a single Ub repeat and a 30 non-coding region of a polyubiquitin gene, wasisolated from wheat (Triticum aestivum) by reverse transcription-polymerase chainreaction (RT-PCR). A PBI sense vector with Ta-Ub2 was constructed and transformedinto tobacco plants. Ub expression in wheat leaves, monitored by semi-quantitativeRT-PCR, responded to drought stress. In transgenic tobacco, determined by proteinoverexpression</p><p>www.elsevier.de/jplph</p></li><li><p>attempt to remove damaged proteins from the cell</p><p>ARTICLE IN PRESS</p><p>Q. Guo et al.1746tailoring of genes has the potential to overcome anumber of limitations in creating drought-toleranttransgenic plants (Umezawa et al., 2006).Ubiquitin (Ub) is a 76-amino acid globular</p><p>protein. As the name implies, Ub is nearlyubiquitous, being present in all eukaryotic speciesexamined. It is also one of the most structurallyconserved proteins yet identied; its amino acidsequence is invariant in all higher plants. Ub is alsounique among plant proteins because it is synthe-sized from fusion-protein precursors. Members ofthe Ub family express either Ub polymers (poly-ubiquitin genes), in which multiples of the 228-bpcoding region are concatenated head-to-tail, orubiquitin extension protein (UbEP) genes, in whicha single Ub-coding region is attached to the 50</p><p>end of another coding region (Callis et al., 1995;Smalle and Vierstra, 2004). These polypeptidesbecome functional after deubiquitination enzymes(DUBs) release them. Free Ubs are attached toappropriate intracellular targets by an adenosinetriphosphate (ATP)-dependent E1-E2-E3 conju-gation cascade (Sullivan et al., 2003; Vierstra,2003). The subsequent addition of Ub moietiesthrough the lysine 48 (K48) residue in Ub resultsin the formation of polyubiquitin chains on thetarget protein. The resulting Ubprotein conju-gates are then recognized and degraded by themultisubunit 26S proteasome with the concomitantrelease of the Ub moieties for reuse. Through thiscycle, the Ub/26S proteasome pathway helpsremove abnormal proteins and thus performs anessential housekeeping function. Ub can also targetcertain normal proteins for breakdown; this path-way provides an important control point byeliminating rate-limiting enzymes and key regula-tory factors and by dismantling crucial signalingnetworks (Vierstra, 2003; Smalle and Vierstra,2004). The inhibition of Ub-dependent proteindegradation can induce cell death program(s) inplants as in animals (Yang and Yu, 2003; Schlogel-hofer et al., 2005; Vaux and Silke, 2005). Data fromyeast and animal studies indicate that in additionto their more traditional roles, the components ofthe Ub/26S proteasome pathway may also haveother functions, some of which may be used byplants. Many of these functions arise from theirability to attach a single Ub or assemble poly-ubiquitin chains using lysines other than K48(Smalle and Vierstra, 2004). Monoubiquitinationcan direct proteolytic targets to the lysosome/vacuole for turnover (Hicke, 2001) or modifytranscription (Bach and Ostendorff, 2003). Anumber of monoubiquitinated proteins have beenidentied, including the H2A and H2B subunits ofthe core nucleosome (Bach and Ostendorff, 2003),in order to maintain cellular function (Fergusonet al., 1990; OMahony and Oliver, 1999; Smalle andVierstra, 2004). In previous experiments (Bachmairet al., 1990; Becker et al., 1993; Conrath et al.,1998; Schlogelhofer et al., 2005), the Ub variant(K48 replaced by arginine (R)) was used as aninhibitor of Ub-dependent protein degradation. Theexpression of UbR48 can cause changes similar tothe inhibition of the proteasome that results in theinduction of various forms of cell death. Theadditional stress causes aggravation of the pheno-type with regard to both the severity and kineticsof symptom appearance (Schlogelhofer et al.,2005). However, there have been very few studiesthus far on the genetic engineering of transgenicplants overexpressing Ub.In this study, Ta-Ub2 was isolated from Triticum</p><p>aestivum using reverse transcription-polymerasechain reaction (RT-PCR). Transgenic tobacco plantsconstitutively expressing the sense RNA of mono-ubiquitin were obtained. The drought resistance oftransgenic plants was investigated. This researchsuggests that Ub may play an important role indrought resistance, and overexpressing monoubi-quitin is an effective strategy to improve droughttolerance in plants.</p><p>Materials and methods</p><p>Plant materials and drought treatment</p><p>Wheat (T. aestivum) and tobacco (Nicotiana tabacum)seedlings were grown in a chamber at 25 1C with a 16/8 hlight/dark cycle (300400 mmol photonsm2 s1) and arelative humidity of 7580%.</p><p>Wheat seeds that had been soaked for 45 h in tapwater were germinated between moistened lter paperfor 24 h and were then arrayed in 10-cm diameter Petridishes (30 seedlings/dish) containing 2 layers of lterpaper wetted with distilled water. After another 24 hof growth, the seedlings were treated with 20%and numerous receptors and transporters at theplasma membrane (Hicke, 2001).Ub is multifunctional (von Kampen et al., 1996),</p><p>and one of its main known functions is to tagproteins for selective degradation by the 26Sproteasome (OMahony and Oliver, 1999; Smalleand Vierstra, 2004). Ub is induced by variousstresses in plants and animals (Fornace et al.,1989; Christensen et al., 1992; Genschik et al.,1992; Sun and Callis, 1997; OMahony and Oliver,1999; Guo et al., 2004). Protein degradation is anormal cellular activity, but an increase in degra-dation in response to stresses can be interpreted asthe result of excessive protein damage and an</p></li><li><p>length, and the fresh weight of seedlings, were investi-</p><p>sequenced with an ABI PRISM 377 DNA Sequencer(Perkin-Elmer, CA, USA); the vector was then introducedinto the Agrobacterium tumefaciens strain LBA 4404 thatwas used for the transformation of tobacco by the leaf-disk method. First, transgenic tobacco plants wereselected on a medium containing 50mgL1 kanamycin.After rooting, the seedlings were transferred to soil andgrown in a greenhouse. After screening with kanamycin,the transgenic plants were detected using genomic PCRand semiquantitative RT-PCR. The transgenic plants(control) carrying the recombinant construct of GUS gene(pBI-GUS) under the control of CaMV 35S promoter and thenopaline synthase 30 termination sequences in the senseorientation and the WT plants were used as controls.</p><p>Semiquantitative RT-PCR</p><p>Total RNA from wheat coleoptiles or tobacco leaveswas treated with DNaseI (RNase-free; Promega) to</p><p>ARTICLE IN PRESS</p><p>Drought tolerance through overexpression of monoubiquitin 1747gated and analyzed statistically.To validate Ub abundance and the physiological</p><p>characteristics (leaf water content and photosyntheticgas exchange parameters) of the transgenic plants,tobacco plants grown for approximately 80 d in plasticpots (14 cm in diameter and 12 cm in height) were used.The natural drought condition was induced withoutwatering for 5 d when the soil water content wasapproximately 50% of the soil saturation moisturecontent. Meanwhile, another group of plants was treatedas the control and were well watered; the soil watercontent of these plants was approximately 75% of the soilsaturation moisture content.</p><p>Isolation of Ta-Ub2</p><p>The total RNA isolated from the wheat coleoptilesusing Trizol reagent (Invitrogen Corporation, Carlsbad,CA, USA) was used for RT-PCR. Briey, 10 mg total RNA wastreated with 10 U RNase-free DNase I (Promega Corpora-tion, Madison, WI, USA) at 37 1C for 15min to removegenomic DNA; RNA was then extracted with phenol/chloroform and nally precipitated in absolute ethanol. A2-mg sample of RNA was denatured at 70 1C for 5min andrapidly ice-quenched; then, 5 mL of reaction buffer, 2mLof 10mM dNTP, 10 U of RNase inhibitor, 1 mL of 10mMoligo-dT primer (50-GACTCGAGTCGACATCGATTTTTTTT-TTTTTTT-30), and 200U of avian myeloblastosis virusreverse transcriptase (Promega) were added. After briefmixing, the transcription reaction was incubated at 42 1Cfor 1 h and terminated at 85 1C for 10min. A degenerate50 primer, namely, WUb1 (50-ATGCA(A/G)AT(T/C/A)TT(T/C)GT(A/G/C/T)AA(A/G)AC-30) that corresponded tothe rst amino acid of Ub, was designed according tothe consensus sequence in other organisms. In order toisolate an Ub-coding region in wheat, a PCR reaction was(w/v, 0.64MPa) or 30% (w/v, 1.32MPa) polyethyleneglycol 6000 (PEG-6000) solution for a further 24 h toinduce drought stress conditions; for the well-wateredcontrol, the seedlings were treated with a uniformvolume of distilled water. The coleoptiles of the wheatseedlings were harvested directly into liquid nitrogen andstored at 80 1C till use.</p><p>Three independent lines of transgenic tobacco plants(T1-2, T1-11, and T1-13) were selected for this study. Inorder to determine the effect of drought stress on seedgermination and seedling growth, the seeds (same ageand water status) of transgenic and wild type (WT) aswell as control tobacco plants (control, transgenic plantscarrying the recombinant construct of the b-glucuroni-dase (GUS) gene but without Ta-Ub2) were sterilized with4% NaClO for 10min and rinsed 56 times in steriledistilled water. They were then sown in Murashige-Skoog(MS) medium containing different concentrations (0, 200,265, 300, and 400mM) of mannitol that was used toinduce drought stress. The germination percentage on MSmedium with 265mM mannitol was investigated. On days35, 60, and 72 after sowing, the growth parameters,including the number of leaves and roots, main rootperformed using the primers WUb1 and B26 (50-GACTCTA-GACGACATCGATTTTTTTTTTTTTTT-30). The PCR productswere cloned into pMD18-T vectors (TaKaRa) and intro-duced into Escherichia coli; 2 transformants were thenselected and sequenced with an ABI PRISMTM 377 DNASequencer (Perkin-Elmer, CA, USA). Two Ub genes, namely,Ta-Ub1 (accession number AY862401) and Ta-Ub2 (acces-sion number AY297059), in wheat were obtained. Ta-Ub2(length, 432bp; Figure 1) contains the last Ub monomerand the 30 non-coding region of a wheat polyubiquitin genethat was selected to construct the expression vector.</p><p>Sense expression of wheat Ub Ta-Ub2 in transgenictobacco</p><p>Ta-Ub2 cDNA in the pMD18-T vectors was digested withSalI and XbaI, subcloned into the pBI121 vectors under thecontrol of the cauliower mosaic virus (CaMV) 35Spromoter and the nopaline synthase 30 terminationsequences. To overexpress Ub in tobacco, Ta-Ub2 wascloned into the pBI121 vector in the sense orientation. Theresulting vector (pBITa-Ub2) was veried by using PCR and</p><p>TM</p><p>Figure 1. Nucleotide acid sequence of the Ta-Ub2 cDNAand its deduced amino acid sequence. The asteriskrepresents the stop codon. The long horizontal arrowsin the 30 untranslated region designate the primers usedfor semiquantitative RT-PCR.</p></li><li><p>CAGAGCACCATC-30 for a-tubulin in wheat. To screentransgenic tobacco plants, the tobacco Actin transcript</p><p>Results</p><p>Characterization of Ta-Ub2 gene</p><p>Using RT-PCR, 2 cDNAs of polyubiquitin geneswere isolated from wheat, namely, Ta-Ub1 (Gen-Bank accession AY862401) and Ta-Ub2 (GenBankaccession AY297059, Figure 1). Ta-Ub2 consists of432 bp nucleotides and a 234-bp open readingframe at positions 1234, encodes an intact Ubmonomer (76 amino acids) and an extra amino acidsequence at its carboxyl terminus. The extra aminoacid is a glutamine residue same as the terminalamino acid sequence reported for a maize poly-ubiquitin (Christensen et al., 1992), and it could beremoved by DUBs after translation (Smalle andVierstra, 2004). Ta-Ub2 was identical (identity was84%) to the last repeat of a wheat polyubiquitin(GenBank accession X56803). Compared with the</p><p>ARTICLE IN PRESS</p><p>Q. Guo et al.1748Determining leaf water content of the transgenicplants</p><p>The leaf water content was calculated as (FWDW)/FW 100%, where FW is the fresh weight and DW is thedry weight after drying the samples at 80 1C for 24 h.</p><p>Analysis of photosynthetic gas exchange</p><p>Completely expanded leaves at identical positions onthe tobacco seedlings were used to estimate the netphotosynthetic rate (Pn), stomatal conductance (Gs), andtranspiration rate (E) with an infrared gas analyzer(CIRAS-2; PP Systems, Hitchin, UK), at a CO2 concentra-tion of 360 mL L1, a saturating light intensity of 800 mmolm2 s1, a gas ow rate of 200mLmin1, and an externalhumidity of 6070%; the temperature inside the leafchamber was 25 1C.</p><p>Statistical analysis was conducted using the dataprocessing system software (Zhejiang University,China).Protein gel blot analysis</p><p>Total protein was extracted from green tobacco leavesof the same age. Protein content was determined by thedye-binding assay according to Bradford (1976). Proteinswere separated by SDS-PAGE on 1014% gradient gel andtransferred to a polyvinylidene uoride membrane(Millipore). Proteins were routinely detected with theUb an...</p></li></ul>

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