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Journal of Plant Physiology 165 (2008) 1745—1755

0176-1617/$ - sdoi:10.1016/j.

Abbreviationzyme; E2, Ub-cleaf stomatal cubiquitin; WT,�Correspond

fax: +86 538 82E-mail addr

1These autho

www.elsevier.de/jplph

Drought tolerance through overexpressionof monoubiquitin in transgenic tobacco

Qifang Guo1, Jin Zhang1, Qiang Gao, Shichao Xing, Feng Li, Wei Wang�

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University,Tai’an, Shandong, 271018, PR China

Received 31 March 2007; received in revised form 8 October 2007; accepted 9 October 2007

KEYWORDSDrought tolerance;Gene expression;Transgenic tobacco;Ubiquitin;Wheat

ee front matter & 2007jplph.2007.10.002

s: E, transpiration ratonjugating enzyme; E3onductance; Pn, net phwild-type tobacco.ing author. Tel.: +86 53842288.ess: wangw@sdau.edu.rs contributed equally

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 proteingel blot analysis, we found higher amounts of Ub–protein conjugates than in control(tobacco carrying a PBI GUS vector without Ta-Ub2) and wild-type (WT) lines.However, free Ub levels did not significantly 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 significantlyhigher under drought stress. Our results indicate that Ub may be involved in theresponse of plants to drought stress and that overexpression of monoubiquitin mightbe an effective strategy for enhancing drought tolerance.& 2007 Published by Elsevier GmbH.

Published by Elsevier GmbH.

e; E1, Ub-activating en-, Ub–protein ligase; Gs,otosynthetic rate; Ub,

8246166;

cn (W. Wang).to this paper.

Introduction

Drought is the primary limitation to wheatproduction 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-creasing evidence has indicated that the molecular

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tailoring 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 globularprotein. As the name implies, Ub is nearlyubiquitous, being present in all eukaryotic speciesexamined. It is also one of the most structurallyconserved proteins yet identified; 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

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 Ub–protein 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 beenidentified, including the H2A and H2B subunits ofthe core nucleosome (Bach and Ostendorff, 2003),

and numerous receptors and transporters at theplasma membrane (Hicke, 2001).

Ub is multifunctional (von Kampen et al., 1996),and one of its main known functions is to tagproteins for selective degradation by the 26Sproteasome (O’Mahony 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; O’Mahony 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 anattempt to remove damaged proteins from the cellin order to maintain cellular function (Fergusonet al., 1990; O’Mahony 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 Triticumaestivum 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.

Materials and methods

Plant materials and drought treatment

Wheat (T. aestivum) and tobacco (Nicotiana tabacum)seedlings were grown in a chamber at 25 1C with a 16/8 hlight/dark cycle (300–400 mmol photonsm�2 s�1) and arelative humidity of 75–80%.

Wheat seeds that had been soaked for 4–5 h in tapwater were germinated between moistened filter paperfor 24 h and were then arrayed in 10-cm diameter Petridishes (30 seedlings/dish) containing 2 layers of filterpaper wetted with distilled water. After another 24 hof growth, the seedlings were treated with 20%

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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.

Drought tolerance through overexpression of monoubiquitin 1747

(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.

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 5–6 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 rootlength, and the fresh weight of seedlings, were investi-gated and analyzed statistically.

To validate Ub abundance and the physiologicalcharacteristics (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.

Isolation of Ta-Ub2

The total RNA isolated from the wheat coleoptilesusing Trizol reagent (Invitrogen Corporation, Carlsbad,CA, USA) was used for RT-PCR. Briefly, 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 finally 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 first 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

performed 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.

Sense expression of wheat Ub Ta-Ub2 in transgenictobacco

Ta-Ub2 cDNA in the pMD18-T vectors was digested withSalI and XbaI, subcloned into the pBI121 vectors under thecontrol of the cauliflower 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 verified by using PCR andsequenced with an ABI PRISMTM 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 50mgL�1 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.

Semiquantitative RT-PCR

Total RNA from wheat coleoptiles or tobacco leaveswas treated with DNaseI (RNase-free; Promega) to

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remove genomic DNA. Reverse transcription was per-formed using the primer 50-GACTCGAGTCGACATCGATT-TTTTTTTTTTTTTTT-30 and Moloney murine leukemia virusreverse transcriptase (Promega) at 42 1C for 60min. Theamplification conditions were as follows: 1min at 94 1C,45 s at 55 1C, and 1min at 72 1C. The cycle was repeated28 times. The Ta-Ub2 gene-specific primers preparedfrom the 30 non-coding region, namely, WUb2 (50-CTGTCAATGGAGCGGCTTCT-30) and WUb3 (50-CCGAACA-TAACGATGCATCA-30) (Figure 1), were used for Ta-Ub2amplification. To detect the expression pattern of Ta-Ub2in wheat coleoptiles, the wheat a-tubulin transcript wasused as an internal positive control for equal cDNAamounts. PCR was carried out by using the primers 50-ATCTGTGCCTTGACCGTATCAGG-30 and 50-GACATCAACATT-CAGAGCACCATC-30 for a-tubulin in wheat. To screentransgenic tobacco plants, the tobacco Actin transcriptwas used as an internal positive control for equal cDNAamounts. PCR was carried out by using the primersNtACT2a (50-CTATTCTCCGCTTTGGACTTGGCA-30) andNtACT2b (50-ACCTG CTGGAAGGTGCTGAGGGAA-30) fortobacco Actin (Yang et al., 2006). The experimentswere independently repeated 3 times under identicalconditions.

Protein gel blot analysis

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 10–14% gradient gel andtransferred to a polyvinylidene fluoride membrane(Millipore). Proteins were routinely detected with theUb antibody (Sigma).

Determining leaf water content of the transgenicplants

The leaf water content was calculated as (FW–DW)/FW� 100%, where FW is the fresh weight and DW is thedry weight after drying the samples at 80 1C for 24 h.

Figure 2. mRNA levels of Ta-Ub2 gene expression underdrought stress. (A) Analysis of expression of the Ta-Ub2gene by semiquantitative RT-PCR (above). The wheat a-tubulin transcript was used as a control for equal cDNAamounts (below). (B) The relative signal density of theTa-Ub2 gene mRNA.

Analysis of photosynthetic gas exchange

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 L�1, a saturating light intensity of 800 mmolm�2 s�1, a gas flow rate of 200mLmin�1, and an externalhumidity of 60–70%; the temperature inside the leafchamber was 25 1C.

Statistical analysis was conducted using the dataprocessing system software (Zhejiang University,China).

Results

Characterization of Ta-Ub2 gene

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 1–234, 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 thesequences obtained from the GenBank database,the deduced amino acid sequence of Ta-Ub2 wasthe same as that of Ubs identified in other plants(Christensen et al., 1992; Callis et al., 1995;O’Mahony and Oliver, 1999). These data suggestthat Ta-Ub2 encodes Ub.

Response of Ta-Ub2 mRNA accumulation todrought stress in wheat

The response of Ta-Ub2 mRNA expression inwheat to drought stress was detected by semi-quantitative RT-PCR (Figure 2) using Ta-Ub2-speci-fic primers (WUb2 and WUb3, Figure 1) constructedfrom the 30 non-coding region of Ta-Ub2. As shownin Figure 2, the Ta-Ub2 transcript levels increased

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Drought tolerance through overexpression of monoubiquitin 1749

slightly in response to 20% PEG (�0.64MPa) in thewheat coleoptiles, but the increase was not verysignificant; the transcript levels decreased inresponse to 30% PEG (–1.32MPa). These resultssuggest that the expression of Ta-Ub2 mRNA may beregulated by drought stress.

Screening of transgenic tobacco plants bysemiquantitative RT-PCR

The sense vector with Ta-Ub2 (pBITa-Ub2) wasconstructed and transformed into tobacco plants.Thirty-two transgenic lines were obtained thatwere detected by genomic PCR after an initialscreening with kanamycin (50mg L�1; data notshown). The tobacco plants were further confirmedas transgenic by semiquantitative RT-PCR using Ta-Ub2-specific primers (WUb2 and WUb3, Figure 1)constructed from the 30 non-coding region of Ta-Ub2. A 135-bp nucleic acid sequence was amplifiedfrom the transgenic plants (partial results; seeFigure 3, lanes 2–6), while no sequence wasamplified from the control and WT plants (Figure3, lanes 1 and 7), indicating that transgenictobacco plants were obtained. Homozygous plantsof 3 lines (T1-11, T1-2, and T1-13; lanes 3, 4, and5 in Figure 3) were used to determine the

Figure 3. Screening of transgenic tobacco by semiquan-titative RT-PCR. (A) mRNA abundance of Ta-Ub2 gene intobacco (above); the tobacco Actin transcript was usedas a control for equal cDNA amounts (below). 1, WT; 2–6,transgenic plants (2–6 are T-1, T-2, T-11, T-13, and T-21transgenic lines, respectively); 7, control; M, marker(DL2000). (B) Relative signal density of Ta-Ub2 geneexpression in transgenic tobacco.

drought tolerance of transgenic tobacco plants(see below).

Abundance of Ub in different transgenicplants

Analysis of Ub abundance by protein gel blot(Figure 4) showed that the overexpression of Ta-Ub2 (a monoubiquitin gene) did not significantlyalter the level of free Ub (8.6 kD, Figure 4B), and nonew protein bands were observed, consistent withthe results of Bachmair et al. (1990). The level ofhigher molecular weight proteins (Ub–protein con-jugates or unprocessed polyubiquitin) in transgenictobacco was higher than that in the WT and controlplants under both the normal water condition andthe drought stress condition.

Figure 4. Abundance of Ub in different transgenictobacco, WT, and control plants. (A) Protein gel blotanalysis of Ub; (B) relative signal density of free Ub(8.6 kD). 1–5 represent WT, control, T1-2, T1-11, and T1-13, respectively, under well-watered conditions; 6–10represent WT, control, T1-2, T1-11, and T1-13, respec-tively, under the drought stress condition.

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Germination and growth status of transgenictobacco

On the MS medium without mannitol (0mM), thegermination rate of the transgenic seeds (T1-2, T1-11, and T1-13) was slightly higher than that ofthe control and WT seeds (Figure 5 legend); thetransgenic seeds germinated completely on the3rd d after sowing, while the control seeds did notgerminate fully until the 4th d after sowing.Drought stress suppressed the germination oftobacco seeds. The impact of drought stress waslesser on the transgenic plants overexpressing Ta-Ub2 than on the control and WT seeds. On the MSmedium containing 265mM mannitol (�0.627MPa)(Figure 5), on the 10th d after sowing, thepercentage of germinated seeds of the transgeniclines varied from 45% to 85%, while that of thecontrols was approximately 17%. The time requiredfor 50% germination was approximately 9–10 d forthe transgenic lines and approximately 11.5 d forthe control and WT seeds. The time taken for 100%germination was approximately 12 d for T1-2, T1-11, and T1-13, while it was nearly 15 d for thecontrol and WT seeds. These results suggest thatthe overexpression of Ub can enhance the germin-ability of the tobacco seeds, particularly underdrought stress conditions.

To determine the growth status under uniformwater conditions, the transgenic tobacco seedsalong with the control or WTseeds were sown in thesame plate (half/half) containing MS medium withdifferent concentrations (0, 200, 300, 400mM) ofmannitol. The number of leaves and roots, length

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of the main root, and fresh weight were observedand analyzed statistically on days 35, 60, and 72(Figure 6) after sowing. The results revealed thatwith regard to the above-mentioned characteris-tics, the 3 transgenic lines grew more vigorouslythan the WT and control samples, indicating thatthe suppression effect of drought stress on thegrowth of transgenic seedlings was lesser than thaton the growth of control and WT plants. Thephotographs of tobacco seedlings growing on MSmedium containing 0 and 200mM mannitol for 21 dare shown in Figure 7A and B. All these resultssuggest that overexpressing Ta-Ub2 from wheat cannot only enhance the tolerance of transgenicseedlings to drought stress but also improve thegrowth status under non-stress conditions.

Water content and photosynthetic gasexchange parameters of transgenic plants

As shown in Figure 8A, under both the well-watered condition and drought stress condition,the water content of the transgenic lines wassignificantly higher than that of the control and WTsamples. Under the well-watered condition, almostno difference was observed among the 3 transgenicplants, the WT plants, and control plants inphotosynthetic gas exchange parameters. Droughtstress induced a decrease in the parameters of allthe tobacco species investigated, particularly inthe net photosynthetic rate (Pn, Figure 8B) andtranspiration rate (E, Figure 8D); however, thedecrease was greater in the WT and control plantsthan in the transgenic plants, indicating thatoverexpressing Ta-Ub2 can improve the physiologi-cal characteristics of the transgenic plants underthe drought stress condition. This suggests that thetransgenic tobacco plants have been endowed withimproved drought tolerance.

Discussion

Ub expression involved in the response ofwheat plants to drought stress

Ub is considered as a stress protein and thus itsresponse to water loss in plants may be a generalstress response (O’Mahony and Oliver, 1999). Theinduction of Ub gene expression under variousstresses was considered necessary to tag thedamaged proteins for selective degradation by the26S proteasome (Ferguson et al., 1990; Garbarinoet al., 1995; O’Mahony and Oliver, 1999). In thisstudy, the expression of Ta-Ub2 increased slightly

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Figure 6. Growth statistics of transgenic and control tobacco plants on MS medium containing mannitol solution (0,200, 300, and 400mM). (A) Number of leaves; (B) number of roots; (C) main root length; (D) FW (fresh weight). The datain (A)–(C) are means7SE, n ¼ 25–30. The values of (D) are means7SE of 3 replications with 10 seedlings each. (A) F(Po0.01): 0 (35 d), 8.787; 200 (35 d), 62.073; 300 (60 d), 49.555; 400 (72 d), 21.04. (B) F (Po0.01): 0 (35 d), 35.105; 200(35 d), 29.403; 300 (60 d), 76.426; 400 (72 d), 37.669. (C) F (Po0.01): 0 (35 d), 23.368; 200 (35 d), 60.401; 300 (60 d),11.232; 400 (72 d), 11.258. (D) F (Po0.01): 0 (35 d), 139.694; 200 (35 d), 76.122; 300 (60 d), 27.778; 400 (72 d), 48.023.

Drought tolerance through overexpression of monoubiquitin 1751

under moderate drought stress (20% PEG, �0.64MPa) but decreased under severe drought stress(30% PEG, �1.32MPa) (Figure 2). This is pos-sibly because moderate drought stress (�0.64MPain this study) induces the accumulation of damagedmacromolecular compounds in the cells that couldtrigger a subsequent adaptation response, e.g., theupregulation of Ta-Ub2 expression. However, se-vere drought stress (–1.32MPa in this study) isdestructive to cellular metabolism, includingthe Ub system, leading to a decrease in the levelof Ta-Ub2 mRNA.

Overexpression of monoubiquitin enhancesdrought tolerance of transgenic tobacco

In the Ub/26S proteasome system, free Ub servesas a reusable recognition signal for selectiveprotein turnover; the resulting ubiquitinated pro-teins are then degraded by the 26S proteasomewith the concomitant release of the Ub moieties forreuse (Pickart, 2001; Weissman, 2001). Proteindegradation increases in response to stress because

of excessive amounts of damaged proteins (O’Mah-ony and Oliver, 1999; Ferguson et al., 1990). Figure4 shows that a higher level of Ub–protein con-jugates in the transgenic plants than in the WT andcontrol plants. This could possibly be an importantreason for transgenic tobacco lines being moretolerant to drought stress.

The overexpression of monoubiquitin couldlead to a more rapid turnover of the Ub pool,resulting in the tagging of more target proteinsunder normal or stress conditions. As shown inFigures 5–7, under both normal water conditionsand the drought stress conditions, the transgenictobacco seedlings grew more vigorously than thecontrol and WT seedlings. The light-saturatedphotosynthetic rate, leaf stomatal conductance,and the transpiration rate of transgenic plants wereall higher than those of the WT and control plantsunder drought stress (Figure 8); this might con-tribute to the excellent growth status of thetransgenic plants.

On the other hand, numerous studies haveindicated that monoubiquitination plays an impor-tant role in plant life (Smalle and Vierstra, 2004).

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Figure 7. Growth status of different transgenic lines under stress conditions. (A–C) Growth on MS medium for 21 d; (A)at normal conditions; (B) under drought stress (200mM mannitol); (C) under salt stress (150mM NaCl); (D–F) growthunder different temperature conditions for 45 d; (D and E) under normal temperature conditions (25 1C), (D) rapidgrowth stage, (E) mature stage, and (F) under low-temperature conditions (10 1C). As shown in (C), the transgenicseedlings (T1-2, T1-11, and T1-13) grew more vigorously than the control plants (WTand control) under salt stress. Undergreenhouse conditions (25 1C), there was no significant difference between the transgenic lines (T1-2, T1-11) and thecontrol plants both at the rapid growth stage (D) and the mature stage (E). After chilling treatment (10 1C) for 45 d (F),the transgenic lines could flower early and had better growth than the control plants.

Q. Guo et al.1752

For example, the addition of a single Ub canpromote or silence genes encoding histone – animportant protein constituent of the DNA andchromosomes of organisms. Monoubiquitination ofmembrane-bound receptors/transporters shuttlesthem via an endosome-mediated trafficking path-way to the lysosome/vacuole for breakdown;conversely, some newly synthesized proteins also

use monoubiquitination to direct transport fromendosomes to the plasma membrane (Hicke, 2001).The overexpression of Ta-Ub2 could afford morefree Ub for monoubiquitination; this is possiblyan important factor contributing to the overexp-ression of monoubiquitin resulting in the enhance-ment of stress tolerance in transgenic tobacco(Figures 5–7).

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−1)

Figure 8. Water content and photosynthetic gas exchange parameters. (A) Water content (%), (B) net photosyntheticrate (Pn, mmol CO2m

�2 s�1), (C) leaf stomatal conductance (Gs, mmol H2Om�2 s�1), (D) transpiration rate (E, mmolH2Om�2 s�1). These parameters were determined at a CO2 concentration of 360 mL L�1, at a temperature of 25 1C,relative humidity of 60–70%, and saturating light intensity of 800 mmolm�2 s�1. Values are means7SE of 3 independentrepetitive experiments from different plants. (A) F (Po0.01): 0 d, 21.801; 5 d, 13.943. (B) F: 0 d (P40.05), 0.294; 5 d(Po0.01), 19.814. (C) F: 0 d (P40.05), 0.306; 5 d (Po0.05), 5.755. (D) F: 0 d (P40.05), 0.075; 5 d (Po0.01), 8.459.

Drought tolerance through overexpression of monoubiquitin 1753

Possible involvement of the Ub/26Sproteasome pathway in multiple toleranceand cross-tolerance in plants

In higher plants, the imposition of abiotic andbiotic stresses often leads to the overproduction ofthe same defense-related secondary metabolites,e.g., reactive oxygen species, which can attack alltypes of biomolecules, including proteins, DNA,carbohydrates, and lipids (Wojtaszek, 1997; Regoliand Winston, 1999; Mithofer et al., 2004). In orderto maintain metabolic stability in cells understress, the timely removal of abnormal anddenatured proteins is crucial (Ferguson et al.,1990; O’Mahony and Oliver, 1999; Kopito, 2000;Kostova and Wolf, 2003; Smalle and Vierstra, 2004).The susceptibility of plants to environmentalextremes has driven the evolution of a wide rangeof stress resistance and tolerance mechanisms,including the Ub/26S pathway (Chen and Zhu,2004; Bohnert et al., 2006; Swindell, 2006). Ubcan be induced by various stresses in plants andanimals (Fornace et al., 1989; Christensen et al.,1992; Genschik et al., 1992; Sun and Callis, 1997;O’Mahony and Oliver, 1999; Guo et al., 2004). Thisstudy showed that the overexpression of mono-ubiquitin enhanced not only drought tolerance(Figures 5–8) but also salt tolerance (Figure 7Aand C) and cold tolerance (Figure 7D–F) in

transgenic tobacco. This suggests that the over-expression of monoubiquitin in transgenic plantscould possibly enhance their tolerance to multiplestresses; this provides new insights into developingplants with enhanced tolerance to various environ-mental stresses.

In addition, it is also suggested that theoverexpression of monoubiquitin and the enhance-ment of the ability of the Ub/26S pathway couldpossibly contribute to the cross-tolerance in plants.As mentioned above, the accumulation of exces-sively damaged proteins may be a universalresponse to injury under almost all types of stresses(Ferguson et al., 1990; O’Mahony and Oliver, 1999;Kopito, 2000; Kostova and Wolf, 2003; Smalle andVierstra, 2004). When its tissue is exposed to amoderate stress condition, the plant is likely toupregulate Ub expression and enhance the functionof the Ub/26S pathway (Figure 2). Plants can utilizecommon pathways and components in the stressresponse; this phenomenon termed cross-toleranceallows plants to adapt/acclimatize to a range ofdifferent stresses (e.g., salt hardiness and coldresistance) after exposure to one specific stress(e.g., drought stress) (Pastori and Foyer, 2002;Mithofer et al., 2004; Lei et al., 2005). Thefunctions of Ub in improving multiple toleranceand cross-tolerance in plants should be furtherstudied.

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Q. Guo et al.1754

Acknowledgments

This research was supported by National NaturalScience Fundation of China (no. 30671259) andNatural Science Foundation of Shandong Province,China (no. Y2003D03).

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