Planta (2007) 225:469–484

DOI 10.1007/s00425-006-0362-5

ORIGINAL ARTICLE

Brassica napus Rop GTPases and their expression in microspore cultures

John Chan · K. Peter Pauls

Received: 30 May 2006 / Accepted: 13 July 2006 / Published online: 9 August 2006© Springer-Verlag 2006

Abstract Androgenesis in plants involves a shift indevelopment that causes cultured microspore cells toform embryos rather than continue to develop pollen.In Brassica napus microspore culture a mild heat stressis used to switch on embryo development. An earlyhallmark of embryogenesis in this system is a symmet-rical division of the nucleus instead of the asymmetricdivision that occurs during pollen formation. ROPGTPases act as molecular switches in a variety ofdevelopmental processes; therefore, the current studywas initiated to examine whether they might beinvolved in androgenesis. Five distinct Rop genes withnucleic acid similarities ranging from 82 to 93% to Ara-bidopsis Rop1 were isolated from B. napus cv Topas. ASouthern blot hybridization with a BnRop sequenceprobe suggested that there are 11–15 ROP gene familymembers in B. napus. RT–PCR reactions with PCRprimers speciWc to BnRop5, BnRop6, BnRop9 andBnRop10 showed that expression of the BnRop5 wasrestricted to pollen but the others were detected inleaf, root, stem and pollen tissue. Pollen-like cellsobtained from 3-day-old cultures by Xow cytometricsorting had BnRop5 transcript levels that were 2.8times higher than in Xow sorted embryogenic microsp-ores. Conversely, the BnRop9 transcript levels were2.5-fold higher in the embryogenic cells than in the pol-len-like cells. The potential involvement of speciWcROPs in early stage microspore culture responses isdiscussed.

Keywords Androgenesis · Brassica · Flow cytometry · Microspore culture · ROP GTPases

AbbreviationsROPs Rho of plantsFDA Fluorescein diacetate

Introduction

Microspore embryogenesis is an example of totipo-tency in plants that involves a switch in developmentfrom pollen maturation to non-zygotic embryo produc-tion. Typically, the embryos that are produced inmicrospore culture contain the gamete chromosomenumber and they develop into haploid plants (Ragha-van 1986). Microspore embryogenesis occurs in a widevariety of species (Reynolds 1997) and is extensivelyutilized by commercial Brassica breeding programs toproduce homozygous lines by chromosome doublingtreatments of the haploids (Powell 1990; Pauls 1996).The developmental process can be induced by stressesapplied to cultured microspores, such as elevated tem-peratures (Keller and Armstrong 1979; Custers et al.1994), ethanol (Pechan and Keller 1989), gamma irra-diation (Pechan and Keller 1989), colchicine (Zhaoet al. 1996), sucrose starvation (Touraev et al. 1996),low temperatures (Sunderland and Dunwell 1974;Kasha et al. 1995) and media pH (Barinova et al.2004).

In responding Brassica microspores the nucleusmigrates to the center of the cell (Telmer et al. 1993).In contrast, during normal pollen development thenucleus is displaced to the cell extremity by vacuolarenlargement (Telmer et al. 1992). Also, in pollen

J. Chan · K. Peter Pauls (&)Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada, N1G 2W1e-mail: ppauls@uoguelph.ca

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development asymmetric nuclear division occurs,resulting in a large vegetative nucleus and a smallergenerative nucleus (Telmer et al. 1993), whereas, sym-metrical nuclear division is observed in inducedmicrospores (Zaki and Dickinson 1991). The earliestmorphological change that occurs in embryogenicmicrospores is the formation of a pre-prophase band ofmicrotubules (Simmonds and Keller 1999). The prep-rophase band marks the position of the new cell wallthat develops between the equally-sized daughter cells.In contrast, the PPB is absent during normal pollendevelopment (Scheres and Benfey 1999).

These observations indicate that alterations in thecytoskeleton play a vital role in reshaping microsporescommitted to the embryogenic pathway. This hypothe-sis is supported by results that demonstrate that themicrotubule depolymerizing agent colchicine caninduce embryogenesis in the absence of heat shock(Zhao et al. 1996). It has been noted that tubulin syn-thesis remains largely unchanged in embryogenicmicrospores compared to non-embryogenic microsp-ores (Cordewener et al. 1996) suggesting that the mor-phogenic changes in embryogenic microspores resultfrom a modiWcation of the organization of the cytoskel-eton and not de novo synthesis of cytoskeletal compo-nents.

ROPs (Rho of plants) are plant-speciWc members ofthe Ras superfamily of GTPases, which includes Wvefamilies: Ras, Rho, Rab/Ypt, Arf and Ran (Zeng andYang 2000). ROPs share 40–60% amino acid identitywith Rho members and greater than 70% amino acididentity with each other. ROPs contain two N-terminaldomains comprising the GTPase domain responsiblefor hydrolysis of bound GTP, two GTP/GDP bindingdomains, an eVector domain that interacts with down-stream targets of ROP, a Rho-insert domain that inter-acts with modiWers of ROP activity and putative serine/threonine phosphorylation sites that are not found inRho proteins (Zheng and Yang 2000). Also, mostROPs contain a CAAL or CAAX polybasic C-termi-nal site that is geranylgeranylated or farnesylated,respectively, and causes the protein to localize to spe-ciWc membranes (Lin et al. 1996). Arabidopsis thalianahas 11 Rop family members (Li et al. 1998). They canbe sub-divided into four groups based on the variableregion and overall sequence similarity: group I(ROP8), group II (ROP9, ROP10, ROP11), group III(ROP7) and group IV (ROP1, ROP3, ROP4, ROP5,ROP6; Li et al. 1998).

ROPs act as molecular switches (Zeng and Yang2000; Fu and Yang 2001) and aVect the cytoskeleton ofplants (Gu et al. 2005). Pollen tube elongation is abol-ished by micro-injecting anti-ROP antibodies into

growing Arabidopsis pollen tubes and suggested thatROP regulates the organization of cortical actin bun-dles in pollen tubes (Li and Yang 2000). This conclu-sion is supported by observations that over-expressionof native Rop1 or constitutive expression of Rop1/Rop5 in Arabidopsis pollen leads to depolarized pollentube growth (Kost et al. 1999; Li and Yang 2000; Che-ung et al. 2003). Also, leaf cell expansion in transgenicArabidopsis over-expressing Rop2 was isotropic,whereas, expression of a dominant negative Rop2resulted in inhibition of cell expansion (Li and Yang2000; Fu et al 2002). The actin bundles in the cells ofconstitutively-active Rop2 transgenics were sub-corti-cal compared to wild-type cells where actin bundleswere cortical (Li and Yang 2000). The similarity ofROPs to RacE in Dictyostelium, which is involved incytokinesis (Larochelle et al. 1996), suggests that theymay be recruited during cell division in plants. Also,because there are no plant homologs to yeast and ani-mal cdc42 or Rho proteins, many researchers have sug-gested that ROPs (and other rac-like small G proteins)are functional substitutes for these proteins involved ingrowth regulation and cellular morphogenesis (Wingeet al. 1997). The multitude of developmental eVectsobserved in transgenic Arabidopsis expressing mutantROPs suggest that ROP signaling controls manyaspects of plant development, including embryo devel-opment (Li et al. 2001).

The purpose of the current study was to determinewhether ROPs are involved in B. napus microsporeembryogenesis. Several cDNAs for ROPs were clonedfrom B. napus pollen and microspore cultures. Theexpression patterns of several of these ROPs in B.napus microspore cultures were determined by RT–PCR and real time PCR. In addition, Rop expressionwas compared in embryogenic and pollen-like cellsXow-sorted from 3-day-old microspore cultures.

Materials and methods

Plant material

The highly embryogenic cultivar Brassica napus cv“Topas” (seeds originally provided by Dr. P.V. Chu-ong, Plant Agriculture Department, University ofGuelph, Ontario, Canada) was used in the study.Plants were established in Sunshine mix (Sun Gro Hor-ticulture Inc., Bellevue, ON, Canada) in a growth cabi-net set to 20°C/16 h light (100 �mol photons m¡2 s¡1)and 18°C/8 dark periods at 60% relative humidity.After the seedlings developed the second internodethey were transferred to 15°C/16 h light (100 �mol

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photons m¡2 s¡1) days and 12°C/8 h dark periods at60% relative humidity. Plants were fertilized with0.4 g/l solution of all purpose 20-20-20 fertilizer (PlantProducts Co., Brampton, ON, Canada) every 2 days.

Microspore culture

Microspores were isolated at the late uni-nucleatestage from whole racemes as previously described (Tel-mer et al. 1992). Isolated microspores were cultured ata cell density of 8.0 £ 104 cells ml¡1 in NLN mediumcontaining 13% (w/v) sucrose (Lichter 1982) in 10 mlvolumes, in Petri plates (150 mm wide, 10 mm deep).The plates were incubated at 25 or 30°C in darkness.

Isolation of total RNA from various plant tissues and reverse transcription to cDNA

Total RNA was isolated from microspores and maturepollen by glass bead disruption (Bio 101, Vista, CA,USA) in Trizol reagent (Invitrogen) following themanufacturers’ instructions. Total RNA was quantiWedby measuring Ribogreen binding (Molecular Probes)with a Shimadzu spectroXuorophotometer (MandelScientiWc, Guelph, ON, Canada) using a 480 nm excita-tion and a 520 nm emission. RNA quality (ribosomalband integrity) was determined by separating 3 �g oftotal RNA by electrophoresis through a 1.5% agarose1X MOPS gel containing 2.2 M formaldehyde andstaining with ethidium bromide. Reverse transcriptionwas carried out using 1 �g of intact total RNA and theRetroscript kit (Ambion, Austin, TX, USA) orSMART RACE kit (Clontech) according to the manu-facturers’ instructions. RNA from leaf, root, earlyembryos and stem tissue was isolated from youngplants by mechanical disruption in a mortar and pestleusing Trizol reagent (Invitrogen).

PCR using degenerate primers

PCR ampliWcation of ROP mature pollen and embryocDNA was performed with degenerate primers 5� Rop(ATGAG(CT)CG(GT)TC(AGT)AAGTT(CT)(AG)T and 3� Rop ((AT)(ACT)ATCA(CT)T(AG)(AG)A(AT)(CG)G(CA)GCA) designed on the basis of tenknown ROP sequences (Rop1-U49971, Rop2-U49972,Rop3-U41295, Rop4-Af031428, GhRac9-S79309,GhRac13-S79308, LjRac1-ZS7s3961, NTRop-Aj222545,Rop1Ps-L19093, O. sativa-Av0299190). PCR was car-ried out over 30 cycles using a Robocycler 96 (94°C,1 min; 58°C, 1 min; 72°C, 1 min; Stratagene) and the600 bp product corresponding to the predicted openreading frame for ROP was puriWed from a 1% agarose

TBE gel, sub-cloned into the PCR cloning vectorTopo 2.1 (Invitrogen) and dideoxy-sequenced on aCEQ 2000 XL (Beckmann Coulter, Mississauga, ON,Canada). The upstream non-coding regions ofBnRop4, BnRop5, BnRop6, BnRop9 and BnRop10 aswell as the downstream 3� UTR of BnRop9 wereobtained by rapid ampliWcation of cDNA ends(RACE) using the SMART RACE kit (Clontech)following the manufacturer’s instructions. Thesequences were complied from at least two separateclones for each cDNA and RACE product, sequencedin both directions.

Isolation of genomic DNA and Southern blot analysis

Total genomic DNA was isolated from young leaf tis-sue using a (modiWed) CTAB method (Doyle andDoyle 1990). BrieXy, 50 �g of genomic DNA wasdigested overnight with the six cutter restriction endo-nucleases BclI and DraI. The digests were separatedon a 5% acrylamide TBE gel, blotted overnight ontocharged nylon membranes (Roche Diagnostic, Laval,QUE, Canada) and probed with digitoxin-labeled,PCR generated BnRop5 synthesized using the PCRDIG Probe Synthesis Kit (Roche Diagnostics).Hybridization was performed using the DIG Easy HybKit (Roche Diagnostics) and Anti-Digoxigenin-APFab fragments (Roche Diagnostics). The hybridizationbands were detected by recording the evolution ofchemiluminescence CDP-Star (Roche Diagnostics) onX-OMAT AR Wlm (Kodak Inc., Rochester, NY,USA).

Phylogenetic analysis

Sequence alignments were performed with Clustal W(Thompson et al. 1994) utilizing the standard defaultparameters for nucleotide and peptide alignments. Thenucleotide alignments of the GTPase ORFs were per-formed in Clustal W and modiWed by adding the 5�

upstream information with Microsoft Word (MicrosoftCorporation, Redmond, WA, USA). The peptidealignments were performed in Clustal W using thedefault parameters and the subsequent Phylip outputwas transformed into a radial phytogram in Treeviewv1.31 (©Roderick D.M. Page 1996).

Flow cytometry

A Coulter EPICS Elite ESP Xow cytometer equippedwith a 15 mW argon–ion laser emitting at 488 nm wasaligned and calibrated using Xuorescent microspheres(DNA-CHECK; Coulter Electronic, Hialeah, FLA,

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USA) for each experiment. The samples were analyzedat a rate of 600 cells s¡1 with a 100 �m Xow cell tip anda sheath pressure of 12 psi. A narrow mask (Coulter,Quartz 0.394R) was used for the forward angle scattercollection which allowed light to scattered from 5° to20° to be collected. Side scatter (90° light scatter) sig-nals were collected using a 488 nm dichroic and 488 nmband-pass Wlter set in front of the photomultiplier tube.Peak signals were recorded for forward scatter, whilean integrated signal was recorded for side scatter. Thegreen Xuorescence intensity signals were collectedbeyond the 488 nm dichroic with a 550 nm dichroic and550 nm band-pass Wlter set.

Two-dimensional histograms of light scatter versusgreen Xuorescence were analyzed using Coul-ter’s®Elite Software (version 4.01). Analysis gates weredrawn around populations of interest and the listmodedata was digitally replayed to obtain light scatter andXuorescent mean channels for the respective popula-tions.

Sorting was performed under the same Xow cyto-metric conditions as described above, with a crystaldrive of 22.1 kHz, at 80% power. The positions of thesort windows were determined using two parameterhistograms of peak forward angle light scatter and logintegrated green Xuorescence. Cells in 2 ml of NLN(8 £ 06 cells ml¡1) were stained with 10 �g of FDA for5 min. Isoton II (Coulter) was used to sort cells intosterile tubes containing NLN. Samples of the sortedcells were reanalyzed by Xow cytometry to characterizepopulation purity and the remaining cells were pelletedby microcentrifugation (1,000g, 5 min), Xash frozen inliquid N2 and stored at ¡80°C for subsequent RNAextraction. Approximately, 1.5 £ 106 cells of each typewere collected in each individual experiment.

Image acquisition

Fluorescent and light images were obtained with aLeica DMRA (Leica, Toronto, Canada) compoundmicroscope equipped with a 100 W mercury lightsource. EpiXuorescence was performed using a 330 nmband-pass UV Wlter set and a 475 nm band-pass blueWlter set (Leica). Images were captured digitally usinga black-and-white CCD camera, Hamamatsu modelC4742-95 (Leica) and OpenLab 3.0 software (Improvi-sion, Guelph, ON, Canada).

Quantitative PCR using gene-speciWc primers

PCR was carried out on microspore derived material(sorted and unsorted) and material derived from vege-tative tissue using real-time monitoring of PCR prod-

uct evolution in a Light Cycler© (Roche Molecular,Laval, QUE, Canada). Primers used to detect BnRop5spanned the 5� untranslated region (5�-TCTGTTC-CTTTCGATCCG-3�) and the open reading frame ofthe gene (5�-TCAGCTCCACGGTATCTTAGT-3�).Similarly, for BnRop9 the primer set spanned the 5� UTR(5�-TAGATCTGAGGGGTTGAATTT-3�) and themain reading frame (5�-GCATAGTGGGT-CAGCTCA-3�). Reactions were prepared followingthe manufacturer’s instructions for BnRop5, BnRop9and S15 transcripts. About 1 �g of mouse total RNA(Ambion, Houston, TX, USA) was added to eachcDNA reaction so that cDNA synthesis eYciencycould be monitored by evolution of mouse S15 cDNA(from the 30S ribosomal subunit S15 gene product)gene accession NMO25544 with 5� primer (5�-AAG-CAGGACGATGAACCA-3�) and 3� primer (5�-CTGGTAACCCGAATGC-3�). Reaction volumeswere 10 �l in glass capillaries. For ORF 5 the cyclingparameters were 10 min denaturation at 95°C followedby 50 cycles (or Xuorescence saturation for the fastestsample) of 95°C for 10 s, 57°C for 10 s, 72°C for 10 s. Asingle data acquisition event measured green Xuores-cence at 83°C between the 95 and 57°C step. The ramprate was 20°C/s between all ampliWcation steps. ThePCR conditions for BnRop9, Rop9 and S15 transcriptampliWcation were 10 min denaturation at 95°C fol-lowed by 95°C for 10 s, 60°C for 10 s, 72°C for 10 s or95°C for 10 s, 58°C for 10 s and 72°C for 10 s, respec-tively. Product purity was assessed for all real-timePCR reactions by melting curve analysis whereby thereactions were heated from 65°C to 95°C at a rate of0.10°C/s with constant data acquisition measuringextinction of Xuorescence.

The cDNA synthesis controls were used to stan-dardize the results obtained from the diVerent experi-mental samples within the same experiment. Theanalyses were performed on RNA isolated from threeseparate microspore cultures, three Xow cytometricsorts of 3-day-old pollen-like cells and 3-day-oldembryogenic cells, and three preparations of vegeta-tive tissue cDNA.

Statistical analysis

Analyses were performed for the microspore timepointdata in SAS for Windows ver 8 (SAS Institute, Cary, NC,USA). ANOVA was performed on the data sets to testthe independent eVects of time, temperature and repeti-tions using the proc glm procedure. Additionally, diVer-ent treatment combinations were compared using theproc univariate procedure. A Shapiro–Wilke statistic wascalculated for each experiment using log-transformed

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data to verify a random distribution of the residuals(P < 0.05) for BnRop5 and BnRop9 data, respectively.For the sorted material, a two-sample t test was per-formed in Microsoft Excel © 97 to compare the means ofinduced and non-induced treatments for both data sets.

Results

BnRop clones

Five unique Rop clones (BnRop4, BnRop5, BnRop6,BnRop9, BnRop10) were obtained by RT–PCR from B.napus pollen-derived mRNA with degenerate primersdesigned from Arabidopsis sequences to amplify thecoding region of a ROP gene. The regions of nucleotidehomology in the Brassica sequences encompassed theentire open reading frame of Arabidopsis Rop1 (660 bp;Fig. 1) and were very similar to this gene sequence. ThediVerences were largely due to randomly scattered singlenucleotide polymorphisms. The similarity of individualsequences to Arabidopsis Rop1 ranged from 82 to 93%.All the sequences that were isolated appeared to befunctional genes. It should be noted that because degen-erate primers were used to obtain the 3� ends there is thepossibility that these regions might contain small errors.However, it is unlikely that any errors are large becauseeach sequence was cloned separately at least twice, thedegenerate sequences encoded all the known Arabidop-sis sequences, only BnROP4 and BnROP5 are identicalfor the 26 bp 3� end and no premature stops or nonsensecodons were detected.

The 5� upstream regions of BnRop4 and BnRop5contained short open reading frames encoding pep-tides of 17 and 16 amino acids, respectively. Theupstream ORFs in BnRop4 and BnRop5 encoded thepeptides MPRCSIEVEHPDNEEEN and MSRN-LIEFEHQEEEQN, respectively. The latter was in thesame reading frame as the main ORF in BnRop5.These two peptide sequences are 58% identical. Inter-estingly, the same regions in both BnRop4 andBnRop5 sequences encoded three-amino-acid peptides(MLD and MVD, respectively) starting nine nucleo-tides from the start of the larger upstream ORFs. Theupstream regions of BnRop6 and BnRop10 also con-tained open reading frames encoding the peptidesMINRVSRGHSYFIVKGEILGFMKDEQLRKHLPRMFSLIKNESWGLEDDQIPS and MPTRDQRMLLLGLRWHLMRNQSFWVPGGGQ for BnRop6,and MQPQSGGKFRPRLNMGERPIANKYREVKMKRTLKRESKSA and MGGQ for BnRop10. Therewere no nested upstream open reading frames in eithergene.

Arabidopsis 5� UTRs

The Wnding that 5� UTRs of four of the Wve Brassicagenes that were sequenced contained small open read-ing frames promoted us to examine the upstream por-tions of a number of the Arabidopsis ROP sequencesavailable from GenBank. Five of the six ArabidopsisROP sequences that were examined also had one ortwo small open reading frames in the Wrst 185 nucleo-tides upstream of the main ROP open reading frame(Fig. 2). Overall, there are striking similarities in sizesand positions of the additional ORFs in the upstreamsequences of the ROP genes in Arabidopsis and Bras-sica. BnRop5 and BnRop4 have ORFs coding for 16and 17 amino acid peptides, approximately 90 nucleo-tides upstream of the main ORF and include very small(three amino acid) peptides nested inside them. Rop1has a very similar structure. BnRop6 and BnRop10also have upstream open reading frames encodingpolypeptides of 52 amino acids and 34 amino acids forBnRop6, and 40 amino acids and 5 amino acids forBnRop10. These open reading frames are arranged ina serial fashion and are much larger than those ofBnRop4 and BnRop5.

Other patterns of open reading frames that wereobserved in the upstream portions of the Arabidopsissequences (uORF) included one short (encoding 3amino acids) and one longer (encoding 20–30 aminoacids) ORF in Rop5 and Rop10; two short (6–10 aminoacids) ORFs in Rop11; one short (4 amino acids) ORFin Rop2 and Rop4; and two long nested sequences, oneof which was in frame and continuous with the mainORF in Rop7 (Fig. 2). Analyses of RNA folding forseveral ROP sequences with upstream open readingframes (using mfold, Mathews et al. 1999) indicatedthat they could include extensive secondary structureswith free energies of ¡39 kCal/mol for BnRop4,¡40.9 kCal/mol for BnRop5, ¡87 kCal/mol forBnRop6, ¡43.6 kCal mol for BnRop9 and ¡55.4 kCal/mol for BnRop10.

ROP small G proteins from Brassica napus

An alignment of the amino acid sequences for the Wveputative B. napus Rops isolated in this study and threepreviously isolated Arabidopsis ROPs (ROP1, ROP3,ROP5; Fig. 3) showed that the sequences are highlyconserved (93–99% identical). Absolute identity wasobserved in the GTPase domains, Rho insert regionand second GTP/GDP binding domain. Conservativesubstitutions occurred in the Wrst GTP/GDP bindingdomain (I114 for V114 and S118 for T118) and in the eVec-tor domain (A52 for S52). The largest variability among

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the sequences occurred in the region between the Rhoinsert region and the second GTP/GDP bindingdomain.

An analysis of phylogenetic relationships amongROPs from B. napus, Arabidopsis, cotton, pea andtobacco (Fig. 4) placed all the B. napus genes into the

Fig. 1 Comparison of the Wve Brassica napus ROP sequenceswith Rop1At of Arabidopsis. The 5� upstream untranslated re-gions are reported for BnRop4, BnRop5, BnRop6, BnRop9 andBnRop10. Lines above the DNA sequences in BnRop4, BnRop5,BnRop6 and BnRop10 indicate upstream open reading frames

(uORFs). Double lines indicate second nested uORFs. Sequencesused to design gene speciWc primers are indicated by boxes,BnRop5 (1,3), BnRop6 (5,7), BnRop9 (2,4), and BnRop10 (6,8).Polymorphisms have white or gray backgrounds

BnRop4 1 ---------------------------------------------------------------------------------------GGCTCATAGGGTTTCTAGTTTTCGGGAGAAAGT BnRop5 1 --------------------------------TTTCTGTCACCTTCCTCTCTCTCTCTCTCTCTCTTTTTGGGCTAACGGGTTTCTCATTTCTGGGACAAAGTCTGTTCCTTTCGATCCG BnRop10 1 -----------------------------------------------------------------------------GAGTACGCGGGGTCTACGAGTCGGGTTGTTCGGGAATGCAGCC BnRop6 1 GGAGTAATGATTAACAGGGACAGTCGGGGGCATTCGTATTTCATAGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGAACAACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC Rop1At 1 ------------------------------------------------------------------------------------------------------------------------ BnRop9 1 ----------------------------------------------------------------------------------------------------------CGATACACCGATTC

consensus GG C GG A AGG GT G T G A GAAA C

BnRop4 34 TCTTTCATTTTTCAAGAATGCCCAGATGCTCGATTGAGGTTGAGCATCCAGACAACGAAGAAGAAAATTAACAAAATCTTTGAGCTTTTCTTTTTATTTTTGGTTTTTCCCCCCTGAAAG BnRop5 89 CAATGTCCAGATGGTTGATTGAATTTGAGCATCAAGAAGAAGAACAAAACTAACAGAGATAGATAGAGAGAGAGCAAAGGATCTGAAGTTCAACGCCTGTTTCAAAGAGATCTGAAAGCT BnRop10 44 CCAATCGGGCGGTAAATTCCGTCCAAGGCTAAATATGGGCGAGAGACCGATAGCGAACAAGTACCGCGAGGTAAAGATGAAAAGGACTTTGAAAAGAGAGTCAAAGAGTGCTTGAAATTG BnRop6 121 AAGAACGAAAGTTGGGGGCTCGAAGACGATCAGATACCGTCCTAGTCTCAACCATAAACGATGCCGACCAGGGATCAGCGGATGTTGCTTTTAGGACTCCGCTGGCACCTTATGAGAAAT Rop1At 1 ------------------------------------------------------------------------------------------------------------------------ BnRop9 15 ATTTCATTCTTCATTGATCGCTTCCTCTCACATACCCGATTTCGTTTAGGGTTAACATTTTCTAGGGTTTTAGAGATAGGCGAAACTGAAATTGAGAAGAAGACGGGTGTTGTTTCCTCA

consensus AT CG A GT AAGAAC C GA GCTCAAA GAGGT GAA A GAGA AAAAAAGA A AGA AAGAAAAAAGGAAAGG TT TAAAA TG T AAAGA T TGAAAA G

BnRop4 154 TATTAACAAATAATCTTCGAGTAGAGAAAATGAGCGCATCTCGGTTCATAAAGTGTGTAACGGTTGGTGATGGAGCTGTCGGAAAAACATGTTTGTTGATTTCTTACACAAGCAACACTT BnRop5 209 CAAATATCTTCGAGCTTTGAGCGGGGACAATGAGCGGCTTCAGATTCGTAAAGTGTGTGACAGTTGGTGATGGAGCTGTCGGAAAAACATGTTTGTTGATTTCTTACACAAGCAACACTT BnRop10 164 TCGGGAGGGAAGCGGATGGGGGGGGGACAATGAGCGCATCGAGATTCGCAAAGTGTGTGACGGTTGGTGATGGAGCTGTCGGAAAAACATGTTTGTTGATTTCTTACACAAGCAACACTT BnRop6 241 CAAAGTTTTTGGGTTCCGGGGGGGGGACAATGAGCGCATCGAGATTCGTAAAGTGTGTGACGGTTGGTGATGGAGCTGTCGGAAAAACATGTTTGCTGATTTCTTACACAAGCAACACTT Rop1At 1 -----------------------------ATGAGCGCTTCGAGGTTCGTAAAGTGCGTGACGGTTGGTGATGGAGCTGTCGGAAAAACTTGTTTGTTGATTTCTTACACAAGCAACACTT BnRop9 135 TAGATCTGAGGGGTTGAATTTTTCGATTTATGAGCGCATCTCGGTTCATAAAGTGCGTGACGGTTGGGGACGGAGCAGTGGGCAAAACATGTCTCCTCATCTCTTACACCAGCAACACTT

consensus TAAAGATGAAAGAT TGGAG GGGGACAATGAGCGCATCGAGATTCGTAAAGTGTGTGACGGTTGGTGATGGAGCTGTCGGAAAAACATGTTTGTTGATTTCTTACACAAGCAACACTT

BnRop4 247 TCCCTACGGACTATGTGCCCACCGTTTTCGATAATTTCAGTGCTAATGTGGTGGTTAACGGAGCCACCGTTAATCTTGGATTGTGGGATACTGCAGGGCAAGAGGATTACAACAGATTAA BnRop5 329 TCCCTACGGATTATGTGCCAACTGTTTTCGATAATTTCAGTGCAAATGTTGTGGTTAATGGAGCCACCGTTAATCTTGGATTGTGGGATACTGCAGGGCAAGAGGATTACAACAGATTAA BnRop10 284 TCCCTACGGATTATGTGCCAACTGTTTTCGATAATTTCAGTGCAAATGTTGTGGTTAATGGAGCCACCGTTAATCTTGGATTGTGGGATACTGCAGGGCAAGAGGATTACAACAGATTAA BnRop6 361 TCCCTACGGATTATGTGCCAACCGTTTTCGATAATTTCAGTGCAAATGTTGTGGTTAATGGAGCCACCGTTAATCTTGGATTGTGGGATACTGCAGGGCAAGAGGATTACAACAGATTAA Rop1At 121 TCCCTACGGATTATGTGCCTACCGTTTTCGATAATTTCAGTGCCAATGTTGTGGTTAATGGAAGCACTGTGAATCTTGGATTGTGGGACACTGCAGGGCAAGAGGATTACAATAGATTAA BnRop9 255 TCCCTACGGATTATGTTCCAACTGTTTTCGATAACTTCAGCGCTAATGTTGTTGTTAACGGAGCCACTGTCAACTTAGGACTCTGGGATACCGCAGGGCAGGAGGATTATAACAGATTGA

consensus TCCCTACGGATTATGTGCCAACTGTTTTCGATAATTTCAGTGCAAATGTTGTGGTTAATGGAGCCACCGTTAATCTTGGATTGTGGGATACTGCAGGGCAAGAGGATTACAACAGATTAA

BnRop4 394 GACCACTAAGCTACCGTGGAGCTGATGTTTTCATATTGGCCTTCTCTCTTATCAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAGCATTACGCGCCTGGTG BnRop5 449 GACCACTAAGCTACCGTGGAGCTGATGTTTTCATATTGGCCTTCTCTCTTATCAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAGCATTACGCGCCTGGTG BnRop10 404 GACCACTAAGCTACCGTGGAGCTGATGTTTTCATATTGGCCTTCTCTCTTATCAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAGCATTACGCGCCTGGTG BnRop6 481 GACCACTAAGCTACCGTGGAGCAGATGTTTTCATATTGGCCTTCTCTCTTATTAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAACATTACGCGCCTGGTG Rop1At 241 GACCACTGAGTTACCGTGGAGCAGATGTTTTCATTTTGGCCTTCTCTCTTATCAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAACATTACGCGCCTGGTG BnRop9 375 GACCCTTGAGTTACCGCGGTGCTGACGTTTTCATCTTGGCCTTCTCTCTCATCAGTAAGGCTAGTTATGAGAATGTCTCCAAGAAGTGGATCCCTGAGCTGACCCACTATGCCCCTGGTG

consensus GACCACTAAGCTACCGTGGAGCTGATGTTTTCATATTGGCCTTCTCTCTTATCAGTAAAGCCAGTTATGAAAACGTCTCCAAAAAGTGGATCCCGGAGTTGAAGCATTACGCGCCTGGTG

BnRop4 514 TCCCCGTCATCCTTGTTGGATCAAAGCTTGATCTTCGAGATGATAAGCAATTCTTCGTCGACCATCCTGGCGCTGTCCCGATTACAACTGCTCAGGGAGAGGAGCTGAGGAAGCTAATAG BnRop5 569 TCCCCATCATCCTTGTTGGATCAAAGCTTGATCTTCGAGATGATAAGCAATTCTTCGTCGACCATCCTGGCGCTGTCCCGATTACAACTGCTCAGGGAGAGGAGCTGAGGAAGCTAATAG BnRop10 524 TCCCCATCATCCTTGTTGGATCAAAGCTTGATCTTCGAGATGATAAGCAATTCTTCGTCGACCATCCTGGCGCTGTCCCGATTACAACTGCTCAGGGAGAGGAGCTGAGGAAGCTAATAG BnRop6 601 TCCCAATCATTCTTGTTGGATCAAAGCTTGATCTTCGAGATGATAAGCAATTCTTCGTCGACCATCCTGGCGCTGTCGCGATTACAACTGCTCAGGGAGAGGAGCTGAGGAAGCTAATAG Rop1At 361 TCCCCATCGTCCTTGTTGGAACAAAGCTTGATCTTCGAGATGATAAACAGTTCTTTATCGACCATCCTGGTGCTGTTCCGATTACTACTGCTCAGGGAGAGGAGCTGAGGAAGCAAATAG BnRop9 479 TCCCTATCGTTCTTGTTGGTACCAAACTAGATCTTAGGGATGACAAACAGTTCTTCGTTGACCACCCTGGTGCTGTACCTATTACCACTTCTCAGGGAGAGGAACTAATGAAGCTAATTG

consensus TCCCCATCATCCTTGTTGGATCAAAGCTTGATCTTCGAGATGATAAGCAATTCTTCGTCGACCATCCTGGCGCTGTCCCGATTACAACTGCTCAGGGAGAGGAGCTGAGGAAGCTAATAG

BnRop4 634 ATGCACCTACTTACATCGAATGCAGTTCCAAATCTCAAGAGAATGTGAAAGCTGTCTTTGACGCAGCCATACGAGTGGTGTTGCAACCGCCTAAGCAGAAGAAGAAAAAGAGCAAAGCGC BnRop5 689 ATGCACCTACTTACATCGAATGCAGTTCCAAATCTCAAGAGAATGTGAAAGCTGTCTTTGACGCAGCCATACGAGTGGTGTTGCAACCGCCTAAGCAGAAGAAGAAAAAGAGCAAAGCGC BnRop10 644 ATGCACCTACTTACATCGAATGCAGTTCCAAATCTCAAGAGAATGTGAAAGCTGTCTTTGACGCAGCCATACGAGTGGTGTTGCAACCGCCTAAGCAGAAGAAGAAAAAGAGCAAAACGC BnRop6 721 ATGCACCTACTTACATCGAATGCAGTTCCAAATCTCAAGAGAATGTGAAAGCCGTCTTTGATGCAGCCATACGAGTGGTGTTGCAACCGCCTAAGCAGAAGAAGAAAAAGAGCAAAGCGC Rop1At 481 GAGCACCTACTTACATCGAATGCAGTTCCAAAACTCAAGAGAATGTGAAGGCGGTGTTTGACGCAGCCATCCGAGTGGTGTTGCAACCGCCAAAGCAGAAGAAGAAGAAGAGCAAAGCGC BnRop9 599 GAGCTCCTTCGTACATCGAGTGCAGTTCAAAATCTCAAGAGAACGTGAAAGGGGTGTTTGATGCAGCGATCAGAGTGGTACTTCAACCTCCAAAGCAGAAGAAAAAGAAGAGCAAGGCAC

consensus ATGCACCTACTTACATCGAATGCAGTTCCAAATCTCAAGAGAATGTGAAAGCTGTCTTTGACGCAGCCATACGAGTGGTGTTGCAACCGCCTAAGCAGAAGAAGAAAAAGAGCAAAGCGC

BnRop4 754 AGAAGGCATGCTCCATCCAGTGA BnRop5 809 AGAAGGCATGCTCCATCCAGTGA BnRop10 764 AGAAGGCATGCTCCATCCAATGA BnRop6 841 AAAAGGCATGCTCCATCCAATGA Rop1At 601 AGAAGGCATGCTCCATTCTATGA BnRop9 719 AAAAGGCCTGCTCCATTTTGTAA

consensus AGAAGGCATGCTCCATCCAATGA

1

2

3 4

5

6

7

8

123

Planta (2007) 225:469–484 475

group IV cluster deWned by Zheng and Yang (2000).The Brassica sequences occurred in two branchesbetween pollen-speciWc Arabidopsis ROP1 and a

branch with Arabidopsis ROP3 and ROP5. The latteris expressed in pollen and vegetative cells (Li et al.1998). The Brassica sequence BnROP9 was a single

Fig. 2 Comparison of the 5� upstream untranslated regions ofseveral Arabidopsis Rops with BnRop4, BnRop5, BnRop6,BnRop9 and BnRop10. Bases in bold represent open readingframes and corresponding translations are indicated. The start co-

dons for the main open reading frames of the genes are boxed.Arabidopsis sequences are designated by Rop and B. napus se-quences begin with BnRop

BnRop9utr -180AAAAAAAAAAAAAAAAACGATACACCGATTCATTTCATTCTTCATTGATCGCTTCCTCTCACATACCCGATTTCGTTTAGGGTTAACATT Rop3utr -180GAAAAAAAAAAAAACGCTTTTGAGATCTTATATTTGAGGTTGTTTCCTCAGGTTTTGTAGATCTGAGGTTGCTATTTTAGAATTAGAGAT Rop4utr -180TTATCTCTCATCGATTTGGCTTGCTACTTTTGAATTTACCCGTGTTCTACTACGATTCGGGATTAGGGTTTTTTCTTGAGTTAGAAAAGA Rop2utr -180CTTCTTCTTTTCCTTCTCTCTTCGATTCTTTTTCGAATTTGGGTGATTCTCAGATTCACAAGCTTTTAGAGCTCGAATTTGATTGGCGAG

M I N E S C N R D I L L T N K Q LM K V V T E T S S L P I N N Y N H H K S F L R T F H H

Rop7utr -180AATGATCAATGAAAGTTGTAACAGAGACATCCTCCTTACCAATAAACAACTATAATCATCACAAAAGCTTCTTAAGAACCTTCCACCACT M V D M S R W L I E F E H Q E E E Q N

BnRop5utr -180TTCTGGGACAAAGTCTGTTCCTTTCGATCCGCAATGTCCAGATGGTTGATTGAATTTGAGCATCAAGAAGAAGAACAAAACTAACAGAGA M L D M P R C S I E V E H P D N E

BnRop4utr -180GCTCATAGGGTTTCTAGTTTTCGGGAGAAAGTTCTTTCATTTTTCAAGAATGCCCAGATGCTCGATTGAGGTTGAGCATCCAGACAACGA M V D Q M P R W S I N

Rop1utr -180CCCCTCTATCTTTTTGGGTAATAGGGTTTCTAATCTCTGGGACAAACTCCAATCGTTTCGAATCCGCAATGCCCAGATGGTCGATCAATA M V N M V E V S E E L A S H C G C F

Rop5utr -180ACATTATACATATCTAGGGTTTTGAGATGGTTAATTGAAAGATAATGGTCGAAGTTTCGGAGGAATTGGCTTCACATTGTGGGTGTTTCT M F G I M L I

Rop10utr -180TATTGAAAGAAAAGTGAAAAAAAAGAGAGTTTAGTTTTTTTAATCATATGGGGTTTATATAGTTTATTACCACACTTCCTCATGTTAATC M Q P Q S G G K F R P R L N M G E R P I A N

BnRop10utr -180GAGTACGCGGGGTCTACGAGGGAATGCAGCCCCAATCGGGCGGTAAATTCCGTCCAAGGCTAAATATGGGCGAGAGACCGATAGCGAACA M F S L I K N E S W G L E D D Q I P S

BnRop6utr -180AAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGAAGACGATCAGATACCGTCCTAGTCTCAACCATAAAC M Q Q T L P S S M L

Rop11utr -180GAACAGAAAAAAAAGCCATAAATTTATGCAACAAACCCTACCTTCTTCTATGCTCTAAAGAGGGTTTATTTATCTTTTACTCCTCTTTTT

BnRop9utr -90TTCTAGGGTTTTAGAGATAGGCGAAACTGAAATTGAGAAGAAGACGGGTGTTGTTTCCTCATAGATCTGAGGGGTTGAATTTTTCGATTTATG Rop3utr -90TCTTTTTTTTTTTTTTCAAAATTTTGTTTGTTGTGATCATACATAGACGTACGTAGCTCCATTTCTGGTGGAGAAGGAAGAAGAAGAGAAATG

M A G I Rop4utr -90TTGTTTCTTGATTCTTGGGAATGGCTGGGATTTGAGGAAGAAGAAGTGCAAAAGGGTTCTTTTTTTGAGTTTGTGAATTTGCTGCGGAGAATG

M S R F Rop2utr -90CTAGAAATGTCGAGGTTTTGAGGAAGAAGCAACCACAAAAGAGGATTGATTTTAAGGGCTTTTTTGTTTGTTTCCGATCTTGCGGCAGACATG

F S S L L N I K P K H I H K I H K H K K I K E I E D K S K E Rop7utr -90TCTCTTCTCTTCTGAACATAAAACCCAAACACATTCACAAAATTCATAAACATAAAAAAATAAAAGAAATTGAAGACAAAAGCAAAGAAAATG BnRop5utr -90TAGATAGAGAGAGAGCAAAGGATCTGAAGTTCAACGCCTGTTTCAAAGAGATCTGAAAGCTCAAATATCTTCGAGCTTTGAGCGGGGACAATG

E E N BnRop4utr -90AGAAGAAAATTAACAAAATCTTTGAGCTTTTCTTTTTATTTTTGGTTTTTCCCCCCTGAAAGTATTAACAAATAATCTTCGAGTAGAGAAATG

R N I K S L Q F Q R L N L Rop1utr -90GAAATATCAAGAGTCTCCAATTCCAAAGGCTAAATCTTTGAGATCTTTTTTTTTATAAATTTCCCTGAAATTAATAAACTTTGAGGGGAAATG

W L P Q V Rop5utr -90GGCTTCCTCAGGTTTAAATCTGAGGTTGATCTCTTTTGTTTTTTGGTAAATTGTGACATATTTTGGCTCGAAGAAGAAGAAGAAGAGGCAATG

T S F I S L L S L S Q S L W F L G V S S L K V V C F W Rop10utr -90ACTTCATTTATCTCTCTATTATCTCTCTCACAATCTCTCTGGTTTTTGGGCGTTTCTTCGTTGAAAGTTGTTTGCTTTTGGTGAGTTAGAATG

K Y R E V K M K R T L K R E S K S A M G G G Q BnRop10utr -90AGTACCGCGAGGTAAAGATGAAAAGGACTTTGAAAAGAGAGTCAAAGAGTGCTTGAAATTGTCGGGAGGGAAGCGGATGGGGGGGGGACAATG

M P T R D Q R M L L L G L R W H L M R N Q S F W V P G G G Q BnRop6utr -90GATGCCGACCAGGGATCAGCGGATGTTGCTTTTAGGACTCCGCTGGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGGGGACAATG

M F V L R S K W Rop11utr -90AAGTAATCTCACTTCATTTATCTCTCTCTCTCTCTCTATTTCTTTTGCTTCCTTTTGGTATTTGCTTTGTATGTTTGTTTTGAGATCAAAATG

BnRop9utr +3-----------------------------------------------------------------------------TAA Rop3utr +3-----------------------------------------------------------------------------TGA Rop4utr +3-----------------------------------------------------------------------------TGA Rop2utr +3-----------------------------------------------------------------------------TGA Rop7utr +3-----------------------------------------------------------------------------TGA BnRop5utr +3-----------------------------------------------------------------------------TGA Bnrop4utr +3-----------------------------------------------------------------------------TGA Rop1utr +3-----------------------------------------------------------------------------TGA Rop5utr +3-----------------------------------------------------------------------------TGA Rop10utr +3-----------------------------------------------------------------------------TGA BnRop10utr +3A----------------------------------------------------------------------------TGA BnRop6utr +3A----------------------------------------------------------------------------TGA

L Q V L Q S S S S V Rop11utr +3GCTTCAAGTGCTTCAAAGTTCATCAAGTGTGTGA-------------------------------------------TGA

123

476 Planta (2007) 225:469–484

member of a branch that occurred closer to the ROP3/ROP5 branch than the main Brassica ROP cluster,which was in accordance with the Wnding that all threesequences had common amino acids at variable posi-tions including: I7, V114, T118, G151 and L197. Theremaining Brassica sequences (BnROP4, BnROP5,BnROP6 and BnROP10) grouped together on a sepa-rate branch that was closest to Arabidopsis ROP1.

Gene copies

A Southern blot of Brassica genomic DNA cut withtwo six-cutter enzymes (BclI or DraI) and probed witha BnRop5 Dig-labeled probe had 11–15 bands largerthan 550 bp (Fig. 5).

BnRop expression studies

PCR primers designed for polymorphic regions inBnRop5, BnRop6, BnRop9 and BnRop10, 5� upstreamregions and the ORFs of these sequences (Fig. 1) wereused to test the occurrence of these forms in cDNAsprepared from diVerent plant tissues (Fig. 6). The ribo-somal RNA bands in the ethidium-stained RNA gels

indicated that the quantity and quality of RNA isolatedfrom the various tissues was equivalent. A Wxed quan-tity of S15 transcript from Mus musculus was added tothe RNA samples to control the eYciency of thereverse transcriptase (RT) reaction and all the samplesproduced a distinct band from PCR reactions withactin primers (although it was slightly less intense inthe pollen sample). BnRop5 expression was restrictedto pollen cells but the BnRop6, BnRop9 and BnRop10transcripts were detected in leaf, root, stem and pollentissue (Fig. 6). The PCR product for BnRop10 was adoublet separated by 100 bp, which may indicate simul-taneous ampliWcation from two diVerent Rop tran-scripts.

To examine the speciWcity of Rop gene expressionduring microspore culture, RT–PCR product synthesiswas monitored by real-time PCR in samples containingGTPase-speciWc primers and RNA from freshly iso-lated microspores or microspores cultured for severaldays at non-inductive (25°C) or inductive (30°C) tem-peratures. Figure 7 indicates that the primers for theseforms were highly speciWc. The BnRop5 primersresulted in a single band of 400 bp only with the clonedBnRop5 sequence but not the cloned BnRop9

Fig. 3 Comparison of the Wve B. napus ROP sequences withROP1, ROP3 and ROP5 of Arabidopsis showing the N-terminalGTPase and eVector domain, two domains responsible for GTP/GDP binding, the Rho-insert region and the C-terminal mem-

brane insert region. Putative phosphorylation sequences SYR,SKK and SSK are shaded in gray. The C-terminal prenylationmotif (P/F) is shaded dark gray; polymorphisms have gray orblack backgrounds

BnROP5 MSGFRFVKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLBnROP6 MSASRFVKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLBnROP10 MSASRFAKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLBnROP4 MSASRFIKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLROP3 MSASRFIKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLROP5 MSASRFIKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLBnROP9 MSASRFIKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPLROP1 MSASRFVKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGSTVNLGLWDTAGQEDYNRLRPL

consensus MSASRFIKCVTVGDGAVGKTCLLISYTSNTFPTDYVPTVFDNFSANVVVNGATVNLGLWDTAGQEDYNRLRPL

BnROP5 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIILVGSKLDLRDDKQFFVDHPGAVPITTAQGEELBnROP6 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIILVGSKLDLRDDKQFFVDHPGAVAITTAQGEELBnROP10 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIILVGSKLDLRDDKQFFVDHPGAVPITTAQGEELBnROP4 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPVILVGSKLDLRDDKQFFVDHPGAVPITTAQGEELROP3 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIVLVGTKLDLRDDKQFFIDHPGAVPITTAQGEELROP5 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIVLVGTKLDLRDDKQFFIDHPGAVPITTVQGEELBnROP9 SYRGADVFILAFSLISKASYENVSKKWIPELTHYAPGVPIVLVGTKLDLRDDKQFFVDHPGAVPITTSQGEELROP1 SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIVLVGTKLDLRDDKQFFIDHPGAVPITTAQGEEL

consensus SYRGADVFILAFSLISKASYENVSKKWIPELKHYAPGVPIILVGSKLDLRDDKQFFVDHPGAVPITTAQGEEL

BnROP5 RKLIDAPTYIECSSKSQENVKAVFDAAIRVVLQPPKQKKKKSKAQKACSIQBnROP6 RKLIDAPTYIECSSKSQENVKAVFDAAIRVVLQPPKQKKKKSKAQKACSIQBnROP10 RKLIDAPTYIECSSKSQENVKAVFDAAIRVVLQPPKQKKKKSKTQKACSIQBnROP4 RKLIDAPTYIECSSKSQENVKAVFDAAIRVVLQPPKQKKKKSKAQKACSIQROP3 KKLIGAPAYIECSSKTQENVKGVFDAAIRVVLQPPKQKKKKSKAQKACSIL ROP5 KKLIGAPAYIECSSKSQENVKGVFDAAIRVVLQPPKQKKKKNKAQKACSIL BnROP9 MKLIGAPSYIECSSKSQENVKGVFDAAIRVVLQPPKQKKKKSKAQKACSIL ROP1 RKQIGAPTYIECSSKTQENVKAVFDAAIRVVLQPPKQKKKKSKAQKACSIL

consensus RKLIDAPTYIECSSKSQENVKAVFDAAIRVVLQPPKQKKKKSKAQKACSIQ

GTPase GTPaseEffector Domain

GDP/GTP Rho insert

Membrane loc. P/FGDP/GTP

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Planta (2007) 225:469–484 477

sequences. Conversely, the BnRop9 primers resultedin a single band of 470 bp only with the cloned BnRop9sequence but not the cloned BnRop5 sequence. Also,in RT–PCR reactions with RNA from control andinduced microspore cultures these primers resulted insingle bands of the appropriate sizes (data not shown).Furthermore, the melting peaks for the products in areal time PCR analysis were in excellent agreementwith predicted values (BnRop5 actual, 83.8°C; BnRop5predicted, 84.1°C; BnRop9 actual, 85.2°C; BnRop9predicted, 85.2°C).

All the RNA samples from cultured microsporeswere of good quality (Fig. 8c) and gave bands of equalintensity with actin primers (Fig. 8d). The amounts ofBnRop5 transcript increased 15.7-fold over day 0 levelsby day 2 in induced cultures (P < 0.05 for means

between 1 day 30°C and 2 day 30°C) but fell back today 0 levels by day 3 and stayed at that level for theremainder of the assay time (Fig. 8a). BnRop9 tran-script levels peaked at day 2 in control cultures but notsigniWcantly. A signiWcant diVerence (P < 0.05) wasobserved between 1 day 30°C and 0 day 30°C levels ofBnRop9 (Fig. 8b). Means comparisons (ANOVA) sug-gested that there were no signiWcant diVerences amongthe timepoint means for both BnRop5 and BnRop9 innon-induced cultures.

Figure 9a shows Xow cytometric proWles of 3-day-old microspores stained with Xuorescein diacetate. Ourgroup has previously shown that this analysis identiWestwo types of cells on the basis of diVerences in greenXuorescence and light scatter and that the diVerencesbetween the two types of cells are suYcient to allow

Fig. 4 Phylogenetic relationships among the Wve B. napus ROPsequences and ROPs from other species. The four groups identi-Wed previously for Arabidopsis ROP sequences (Zhi-Liang andYang 2000) are shown. Arabidopsis (ROP), Brassica campestris(B), Brassica napus (Bn), Gossypium hirsutum (Gh), Lotus japo-nicus (Lj), and Nicotiana tabacum (Nt). Genebank accessionnumbers for the sequences are as follows: ROP1 (AF085480),

ROP2 (V45236), ROP3 (AF115466), ROP4 (AF115472), ROP5(AF115473), ROP6 (AF115470), ROP7(AF115469), ROP8(AF156896), ROP9 (AF115474), ROP10 (AF079486), ROP11(AF079485), (BRACl (AF042330), BnROP4 (AY168614),BnROP5 (AY168615), BnROP6 (AY168616), BnROP9(AY168617), BnROP10 (AY168618), GhRAC9 (S79309),GhRAC13 (S79308), LjRAC1 (Z73961), NtROP (AJ222545)

BnROP6

BnROP10 BnROP5

ROP3ROP5

BnROP9

ROP1

NTROP

LjRAC1

ROP6BrRAC1

ROP2

ROP4

ROP8

ROP7GhRAC9 GhRAC13

ROP10

ROP11

ROP9

0.1

BnROP4

III

I

II

IV

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478 Planta (2007) 225:469–484

sorting by the third day of culture (Schulze and Pauls1998). Populations of microspores that were enrichedfor the less-Xuorescent, lower light scatter-type cells(Figs. 9b, 10a) or the more-Xuorescent cells (Figs. 9c,10d) were obtained by Xow sorting. Microscopic analy-sis of the sorted cells showed that the less-Xuorescentcells were round and had a clear cytoplasm (Fig. 10b).This population of cells has previously been shown tobe embryogenic (Schulze and Pauls 1998). The cells inthis population had two equally-sized nuclei after stain-ing with Hoescht (Fig. 10c). The cells with higher levelsof green Xuorescence sorted from the induced cultureswere oblong with a dense cytoplasm (Fig. 10e). AfterHoescht staining these cells was shown to contain threenuclei, including one diVuse vegetative nucleus andtwo dense generative nuclei (Fig. 10f). Flow cytometricre-analysis of the sorted cells conWrmed that the sortingprocess was eVective. The sorted population of round,embryogenic cells was more than 96% pure (Fig. 9b)and the population of pollen-like cells was 88% pure(Fig. 9c).

Real time PCR analyses of Rop transcript levels inmicrospore populations enriched for pollen-like andembryogenic cells from 3-day-old cultures (asdescribed above) showed that the BnRop5 transcriptlevel was 2.8 times higher in the pollen-like microsp-ores than the embryogenic microspores (P < 0.05;

Fig. 11a). Conversely, the BnRop9 transcript levelswere 2.5-fold higher in the embryogenic cells than thepollen like cells (P < 0.05; Fig. 11b). However, it isclear from the mixed cultures (Fig.8) that the relativelevels of speciWc BnROPs in the microspores werequite dynamic and additional analyses of pure culturesof embryogenic cells at earlier and later time pointswould be beneWcial. Unfortunately, it was not possibleto identify embryogenic cells at earlier time points andolder cultures were very diYcult to sort because theytended to occlude the Xow cell tip.

Discussion

ROP GTPases in Brassica

RT–PCR cloning with Brassica napus pollen- or micro-spore-derived mRNA and primers based on Arabidop-sis ROP sequences resulted in the identiWcation of Wve

Fig. 5 Brassica napus Rop gene family. A Southern blot of B. na-pus cv. Topas genomic DNA cut with the six-cutter enzymes BclI,DraI or BclI + DraI was probed with BnRop5

Bcl

I / D

raI

Bcl

I

Dra

I

Sta

nd

ard

s

564

20272322

4361

65579416

Fig. 6 Expression of B. napus Rops in various tissues. RT–PCRproducts with primers for a BnRop5, b BnRop6, c BnRop9, dBnRop10 and e actin. g Total RNA separated with a 1.5% aga-rose formaldehyde denaturing gel and stained with ethidium bro-mide. f Positive control for cDNA synthesis eYciency obtained byspiking the RNA samples with S15 (a mouse gene) mRNA beforeperforming the reverse transcriptase reaction and amplifying withprimers for S15

a) BnRop5

c) BnRop9

f) S15

g)Total RNA

e) Actin

Lea

f

Ro

ot

Ste

m

Po

llen

b) BnRop6

d) BnRop10

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Planta (2007) 225:469–484 479

new Rop GTPase sequences from B. napus. Comple-mentation of a yeast vesicular transport mutation by aB. napus clone encoding a small GTP-binding proteinwas previously reported (Park et al. 1994) but thissequence is not a ROP due to the lack of the SYR andSSR phosphorylation sites and a diVerent eVectordomain. The protein is more closely related to the Rabfamily of small G proteins which are responsible forvesicular traYcking in the cell. An EST sequencenamed Brac1 was isolated from Brassica rapa(AF042330). From a comparison of this sequence toArabidopsis and Brassica Rops it is likely that thissequence is also a Rop. No additional reports of smallGTPases from B. napus exist. A word search withGTPase against the TIGR Brassica oleracea databaseretrieved six sequences with homology to ArabidopsisRas-like GTPases but they group in the Rab subclassof Ras. Therefore, the current report is the Wrstdescription of Rop genes in B. napus .

The sequences retrieved from B. napus were verysimilar to Arabidopsis sequences (from 92 to 93%homologous). This result may reXect the fact that Ara-bidopsis sequences were used to design the degenerateprimers used for PCR cloning, but also likely reXectsthe relatedness of the two species (Cavell et al. 1998).

Multiple copies and roles for Rop GTPases

The Rops in B. napus exist as a multigene family. This isin agreement with reports of multigene Rho GTPasefamilies in other plants (Yang and Watson 1993; Delmeret al. 1995; Winge et al. 1997; Li et al. 1998; Kawasakiet al. 1999; Trotochaud et al. 1999; Schiene et al. 2000;Valster et al. 2000). The current estimate of 11–15 copies

of Rops from the Southern blot data seems realisticgiven that Arabidopsis has 11 genes. However, becauseof the allo-tetraploid nature of B. napus, it is possiblethat additional copies exist that were not detected withthe BnRop5 probe used in the current study. The genesidentiWed in the current study clustered with members ofgroup IV in Arabidopsis. Members of this group have ashort variable C- terminus involved in membrane locali-zation (Zheng and Yang 2000).

GTPase proteins

All of the sequences that were isolated in the presentstudy appear to be functional genes since they possessopen reading frames encoding 197 amino acid polypep-tides that are very similar to Rop1 from Arabidopsis.All of the sequences possess two eVector domains,

Fig. 7 a, b SpeciWcity of PCR primers for BnRop5 and BnRop9.RT–PCR products obtained with BnRop5 primers (a, boxes 1 and3, Fig. 1) and BnRop9 primers (b, boxes 2 and 4, Fig. 1 andBnRop5-Topo2.1 and BrRop9-Topo2.1 cloned templates,respectively

Bn

Ro

p5-

To

po

2.1

Bn

Ro

p9 -

To

po

2.1

a) BnRop5 primers

b) BnRop9 primers

Fig. 8 a-d BnRop5 and BnRop9 transcription in freshly isolatedmicrospores and microspore cultures over 5 days. The cultureswere given a mild heat stress (30°C) to induce embryogenesis orkept at 25°C (control conditions). Transcript levels are given asrelative values to levels measured in freshly isolated 0 day mi-crospores. c RNA integrity as determined by staining total RNAseparated with a 1.5% agarose formaldehyde gel with ethidiumbromide. d Controls for cDNA synthesis obtained by amplifyingB. napus actin from cDNA preparations. Bars represent § SE ofthe treatment samples calculated for three separate experiments.Bars with the same letter were not signiWcantly diVerent(P < 0.05) according to a LSD

0.4

0.6

0.8

1

1.2

1.4

1.6

d) Actin

c) Total RNA

tn

uo

ma tpircs

narT

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480 Planta (2007) 225:469–484

reXecting the broad scope of downstream eVectors thatare targeted by ROPs. These include the RHO insertregion and the conserved small GTPase eVectordomain. The RHO insert region is unique to the Rhosubclass and consists of a stretch of 9–10 amino acidsthat is necessary for interaction with other proteinscalled RICs (Rho-interacting CRIB-domain containingproteins; Zheng and Yang 2000; Wu et al. 2001; Fuet al. 2005; Gu et al. 2005). The conserved smallGTPase eVector domain may interact with cdc42/Ran-type eVectors such as phosphatidylinositol monophos-phate kinase (PtdIns P-K; Kost et al. 1999) or the cal-lose synthase complex (Hong et al. 2001). The RHOinsert regions of the Wve Brassica sequences were100% identical with each other and only diVered fromthe Arabidopsis ROP1/ROP3/ROP5 genes by one con-servative substitution (I130!V130). Similarly, the WveBrassica conserved small GTPase eVector domainswere 100% identical and only diVered by one aminoacid (S52!A52 ) from ROP1 of the three Arabidopsis

sequences to which they were compared. The similari-ties among the eVector regions suggest that all theseROPs have similar downstream targets.

All of the Brassica sequences possess the SYR puta-tive serine/tyrosine phosphorylation site that distin-guishes ROPs from other small G proteins (Zheng andYang 2000). In addition, the sequences identiWed in thecurrent study have the putative SKK serine/threoninephosphorylation site at residue 98–101 (Zeng and Yang2000), which is also characteristic of the group IV clas-siWcation. A third potential phosphorylation site (SSK)occurs in the second GDP/GTP binding domain. Thephosphorylation sites may be targets for receptor-likeserine/threonine kinases like CLAVATA1 (Troto-chaud et al. 1999).

All of the Brassica sequences have two GTPasedomains that are identical to those in the ArabidopsisROP1, ROP3 and ROP5 sequences. A high level ofresidue conservation was also observed in the twoGTP/GDP binding domains. The second GTP/GDP

Fig. 9 Flow cytometric pro-Wles of 3-day-old microspore cultures stained with Xuores-cein diacetate. a Heteroge-neous 3-day-old microspore cultures. b Re-analysis of embryogenic Xow sorted cells (lower population in Fig. 8a). c Reanalysis of sorted pollen-like cells (upper population in Fig. 8a)

b) c)

.

Gre

en F

luo

resc

ence

Forward Scatter

.

a)

Forward Scatter

Forward Scatter

Fig. 10 Microscopic images of Xow sorted microspores from 3-day-old cultures. a, b, c Images of embryogenic cells. d, e, f Pollen-like microspores. a, d Green Xuorescence after FDA staining. b,

e Cellular morphology revealed by phase contrast microscopy. c,f Nuclei stained with Hoescht. Scale bars values are 50 �m (a, d)and 10 �m (b, c, e, f), respectively

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Planta (2007) 225:469–484 481

binding domain region in BnROP9 had the conserva-tive substitution (S118!T118) that may have little or noeVect on the function of the domain. RAC/ROP Guan-ine exchange factors (RhoGEFs) catalyze theexchange of GDP for GTP at these sites to activate theGTPase and may act to connect the signal from recep-tor kinases to ROPs (Berken et al. 2005; Kaothienet al. 2005).

A major diVerence between most of the ROPs iso-lated from Brassica (BnROP4, BnROP5, BnROP6,BnROP10) compared to group IV Arabidopsissequences is that they contain the farnesylation motifCAAX (cysteine, two aliphatic amino acids and anyamino acid except for L) instead of the CAAL geranyl-geranylation motif in their C-termini. This suggeststhat these B. napus ROPs are localized to membranesystems predominantly through farnesyl modiWcations.However, the BnROP9 sequence was like the Arabid-

opsis Rop1 sequence, in which it contained the CAALgeranylgeranylation motif. Therefore, the pattern of C-terminus modiWcation in particular isoforms does notappear to be conserved among species since the pollen-speciWc Arabidopsis ROP1 contains the CAAL motif,whereas the pollen speciWc form in Brassica (BnROP5)contains a CAAX motif. It is also important to notethat localization of ROPs to membrane systems hasbeen shown to occur independently of an isoprenyla-tion domain as in the case of ROP9, ROP10 andROP11 in Arabidopsis (Lavy et al. 2002).

DiVerential expression of Rops

Like other studies (Li et al. 1998), the current resultsshow that ROPs are diVerentially regulated in diVerenttissues. For example, BnRop5 expression wasrestricted to pollen, whereas BnRop9 mRNA wasfound in all the tissues that were sampled. Therefore,the former is most similar to Arabidopsis Rop1,whereas the latter had an expression pattern like Ara-bidopsis Rop3 or Rop5. Given the multiple forms ofROPs in plants, evidence for diVerential subcellularlocalizations of diVerent forms (BischoV et al. 2000;Ivanchenko et al. 2000) and diVerential interactionsthat have been observed between ROPs and theirunique class of eVectors called RICs (Wu et al. 2001),there is potential for an extremely rich array of signalsthat can be transmitted by these molecular switches.

5� UTRs and gene regulation

Only a small percentage of all eukaryotic genes arethought to contain uORFs (less than 10%) but two-thirds of oncogenes and genes controlling cellulargrowth and diVerentiation appear to posses them. It isthought that they provide a mechanism for gene regu-lation that is largely based on inhibition of translationof the main ORF (Ng et al. 2004; David-Assael et al.2005; Wiese et al. 2005). Perhaps, these sequences areinvolved in regulating mRNA abundance in particulartissue types because BnRop5 and Rop1 accumulatespeciWcally in pollen. In contrast, BnRop9 and Rop3,which do not have any upstream open reading frames,are found in all tissues (Li et al. 1998). However, wealso observe that BnRop6 and BnRop10 containedupstream open reading frames and are expressed in alltissues as well. BnRop4, BnRop5 and BnRop10showed extensive secondary structure in the upstreamregions. In contrast, the RNA folding predictions forthe BnRop9 and Rop3 upstream regions showed littlesecondary structure. Perhaps, the folding in the pollen-speciWc ROP mRNA allows it to survive storage in

Fig. 11 Expression levels of BnRop5 (a) and BnRop9 (b) mRNAin 3-day-old sorted embryogenic and pollen-like cells. Data is pre-sented as number of copies of target transcript per 50 picogramsof reverse-transcribed total RNA. c RNA integrity determined bystaining total RNA separated with a 1.5% agarose formaldehydegel with ethidium bromide. d Controls for cDNA synthesis ob-tained by amplifying B. napus actin from cDNA preparations.Means § SE are shown, n = 3

c) Total RNA

d) Actin

a) BnRop5

b) BnRop9

Tra

nsc

rip

t am

ou

nt

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rip

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0

100000

200000

300000

400000

500000

0

4000

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482 Planta (2007) 225:469–484

these specialized cells (Hulzink et al. 2002). The pres-ence and absence of uORFs in members of the highlyrelated ROP gene family might provide an interestingmodel system to explore the roles of these elements ingene regulation.

A model for ROP GTPase function in microspore culture

In many organisms ROPs bring about their morpho-genic eVects by modifying the organization of the cyto-skeleton (Zheng and Yang 2000; Gu et al. 2005). Thecurrent results indicate that the mild heat stress thatcauses the shift in development in cultured B. napusmicrospores toward embryo production is accompa-nied by signiWcant accumulations of at least two ROPmRNAs, namely BnRop5 and BnRop9. At the cellularlevel this transition is accompanied by the develop-ment of a small number of embryogenic cells (approxi-mately 1–10% of the cells in culture) that swell, take ona round appearance and undergo a symmetricalnuclear division (Nitta et al. 1997; Schulze and Pauls1998; Simmonds et al. 1999). Embryogenic cellsdevelop a pre-prophase band of microtubules that deW-nes the symmetrical plane of division (Simmonds et al.1999) but non-responding cells, that continue pollendevelopment, do not form the pre-prophase band. Fur-ther evidence for the involvement of the cytoskeletonin this developmental transition is the Wnding that col-chicine (which binds to tubulin) can also induceembryogenesis in these cultures (Zhao et al. 1996).

In most references to the interaction of GTPaseswith the cytoskeleton, actin microWbers are consideredto be their downstream targets. However recently, agene for a protein with homology to a family of pro-teins that mediate cytoskeletal re-organization inresponse to extracellular signals and contains a domainfor interacting with RAC-like GTPases was identiWedusing an Arabidopsis mutant that aVects cell shape(Qui et al. 2002). This protein appears to regulate theorganization of the microtubule array during inter-phase and the authors suggest that, since ROPs are theonly set of small GTPases in plants, they may interactwith both types of cytoskeletal elements. The small Gprotein Ran has also been shown to be involved in theself-organization of microtubule asters in Xenopus eggextracts (Ohba et al. 1999).

Although it was diYcult to observe the measure-ments made with the whole cultures it was quite clearin the measurements made with the sorted cells thatthe transition to the embryogenic state was accompa-nied by a shift in the form of GTPase being synthesizedby the cells. In particular, the sorted embryogenic cells

were enriched for BnRop9 mRNA, which might beconsidered a vegetative form based on its occurrence inleaf, root and stem tissue. In contrast, the sorted pol-len-like cells had high levels of BnRop5, which was theform restricted to pollen cells in the tissue surveyexperiments. Large accumulations of pollen speciWcforms of ROPs have been observed previously in Ara-bidopsis (Li et al. 1998). We propose that the shift inthe cultured cells that begin to swell and become roundafter receiving the inductive stimulus to the vegetativeform of ROP (BnROP9) is at least symptomatic, if notcausative, of the commitment to the embryogenic path-way.

In normal microspore maturation the Wrst asymmet-ric division is mediated by the formation of an oV-cen-ter set of microtubules anchored to a collection of actinmicroWlaments at a plasma membrane (PM) focus andstretching to the nucleus. The asymmetrical or polardevelopment of the actin could be mediated byBnROP5 as described for other polar systems, includ-ing pollen tip growth (Li et al. 1999). Stress mightbreak a connection between the PM/actin/microtubule/nucleus complex and allow the nucleus to take a posi-tion at the center of the cell. Once the fate of the cellhas been reset toward the embryogenic pathway itwould still require ROP activity for nuclear divisionand remodeling of the cell. For example, the cell swell-ing that is observed in the embryogenic cells has beenobserved in Arabidopsis cells expressing a constitutivemutant of ROP (Fu et al. 2002). Also, ROP has beenshown to be a regulatory subunit of 1,3-�-glucan syn-thase, required for callose deposition in the cell plateArabidopsis (Hong et al. 2001). These changes mightbe mediated by a vegetative form of ROP likeBnROP9.

Cell remodeling initiated by ROPs might be medi-ated by the class of Rop-interacting CRIB containingproteins known as RICs. SpeciWc ROP/RIC interac-tions in these cells have been implicated in the controlof cellular morphology in both pollen (Gu et al. 2005)and leaf pavement cells (Fu et al. 2005. It is possiblethat similar interactions, between BnROP5 orBnROP9 and speciWc RICs, could determine if themicrospore cell undergoes isotropic cell expansion(embyrogenesis) or normal pollen formation.

Acknowledgments Thanks to Angela Hill for assistance withpreparation of the manuscript, Dr. Laima Kott for her adviceregarding microspore culture, Dr. Ken Kasha for assistance inobtaining the epiXuorescent images and Dietmar Scholz for assis-tance with the growth facilities. Special thanks to Janice Brazolotfor providing technical support for the Xow cytometry. The workwas supported by a grant from the Natural Sciences and Engi-neering Research Council of Canada to KPP.

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