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NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco Blackwell Publishing Ltd

Lu Wang*, Fang-Lei Liao*, Li Zhu, Xiong-Bo Peng and Meng-Xiang Sun Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China

Summary Author for correspondence: Meng-Xiang Sun Tel: +86 27 68756170 Fax: +86 27 68756010 Email: [email protected] Received: 5 December 2007 Accepted: 18 February 2008

• The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. • A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. • In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. • The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development. Key words: ARF-GEF, cell dividing pattern, embryogenesis, GNOM, Nicotiana tabacum (tobacco), pollen tube. New Phytologist (2008) 179: 81–93 © The Authors (2008). Journal compilation © New Phytologist (2008) doi: 10.1111/j.1469-8137.2008.02444.x

Introduction Embryogenesis and postembryonic development in plants depends greatly on polar auxin transport, which requires the proper asymmetric localization of auxin influx and efflux carriers. The genetic and molecular regulation of embryo development has been extensively studied in Arabidopsis thaliana. Studies have revealed that in this model plant, trafficking and localization of the putative auxin efflux facilitator PIN1 (PIN-FORMED1) are influenced by GNOM (Mayer et al., 1991; also called EMB30: Meinke, 1985; Baus et al., 1986; Shevell et al., 1994; Busch et al., 1996; Steinmann et al., 1999), a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small G proteins. Several studies have examined the function of GNOM and its functional mechanism through mutant analysis (Mayer et al., 1993). In the gnom mutant, *These authors contributed equally to this work.

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zygotes tend to divide symmetrically, and typically both the radial and apical-basal patterns of the embryos are interrupted. During early embryogenesis in these mutants, severe defects in cell–cell alignment, cell division and expansion, and cell wall components have also been observed, resulting in misshapen embryos, having lost their apical-basal axis. In some weak mutants, embryogenesis is not seriously interrupted, but various abnormalities arise during postembryonic development. For example, root growth is inhibited. In particular, the root tip grows much more slowly and the stem cells differentiate prematurely. Meanwhile, irregular and discontinuous venation with the formation of clustered or scattered tracheary elements has been observed. Furthermore, according to Koizumi et al. (2005), the rosette leaves produced by the van3 emb30-7 double mutant are similar to those produced by the van3 mutant, which suggests that the concentrated vascular pattern induced in the gnom/emb30 mutant is suppressed by the van3 mutation. In the work of Sieburth et al. (2006), the

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phenotype of double mutant of sfc and a weak gnom allele gn4577 suggested mutual suppression of each single mutant phenotype. This revealed that the SFC ARF-GAP might function in a pathway opposing that of the GNOM ARF-GEF. Recently, Fischer et al. (2006) reported that a combined action of the auxin influx carrier AUX1, ETHYLENEINSENSITIVE2 (EIN2), and GNOM genes provides the vectorial information for planar polarity in Arabidopsis. Specifically, an auxin concentration gradient is virtually nonexistent in aux1 ein2 gnomE/B roots in which locally applied auxin can coordinate hair positioning. This suggests that AUX1, EIN2, and GNOM function together upstream of Rho-of-Plant (ROP). Accumulated data have revealed that GNOM plays a critical role in pattern formation during embryogenesis and in postembryonic development. Meanwhile, the Arabidopsis and rice genome sequencing projects have identified other members of the GNOM subfamily. GNL1, which is closely related to GNOM, is located on A. thaliana chromosome 5. Recently published evidence on Arabidopsis indicates that GNOM and GNL1 share one common, essential function in ER-Golgi trafficking, whereas only GNOM has another, unique essential function in endosomal recycling. GNL1 activity is required for the integrity of Golgi stacks and for COPI-coated vesicle formation. Seedlings of gnl1 were slightly smaller with reduced lateral root number. In some lines with strong phenotypes, the adult plants were bushy and stunted with 30% ovule abortion. Floral organs also did not open fully and leaf expansion was reduced (Richter et al., 2007; Teh & Moore, 2007). However, how the gene is involved in these developmental processes, especially cellular developmental events, is not reported in detail. Recently, we cloned NtGNL1 in tobacco. Unlike GNL1, NtGNL1 is likely brefeldin A (BFA)-sensitive. The use of RNA interference (RNAi) to knock down the expression of this gene showed that NtGNL1 plays an important role in embryo development and postembryonic growth. Partially restrained phenotypes were also observed in pollen germination and tube growth, indicating that NtGNL1 is involved not only in embryonic development and root growth, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.

Materials and Methods Plant materials Nicotiana tabacum L. cv. Petite Havana SR1 plants were grown under 16 h of daylight at 25°C in a glasshouse or axenically in incubators. Isolation of full-length NtGNL1 A cDNA pool was synthesized using the SMART-RACE System (Clontech, Palo Alto, CA, USA) according to the manufacturer’s

New Phytologist (2008) 179: 81–93

instructions. Briefly, an aliquot of total RNA (approx. 5 mg) was reverse-transcribed using a SMART cDNA synthesis primer. GN3 L, which served as the 3′ RACE primer (5′-CAC AGG TCA AGA AGA AGA TGA CGG AGG-3′) in combination with the CA and RA primers provided in the kit, was used for RACE PCR. The primary PCR conditions were 95°C for 15 s, 65°C for 25 s, and 72°C for 5 min (25 cycles), while the secondary PCR conditions were 95°C for 15 s, 67–62°C (touchdown, reduction of 0.2°C per cycle) for 25 s, and 72°C for 5 min (25 cycles). LA-Taq polymerase and the dNTPs were purchased from Takara. The 5′ RACE was performed in nearly the same way, except that oligo-dT was used as the reverse primer (i.e. CA and RA were used, and GN5 L served as the 5′ RACE primer (5′-CGG TTG TTG CGA ATG AAA TCT TCC TCC-3′)). The PCR conditions were altered as follows: (primary PCR, 25 cycles) 95°C for 15 s, 65°C for 25 s, and 72°C for 5 min; and (secondary PCR, 35 cycles) 95°C for 15 s, 67–60°C (touchdown, reduction of 0.2°C per cycle) for 25 s, and 72°C for 5 min. The amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). At the 3′ and 5′ untranslated regions of each RACE product, two new primers were designed to validate the integrity of the cDNA sequence and to amplify the full-length (i.e. genomic) sequence (TGU: 5′-AAC TAT GAT GGG GTG CCT TAA TCA GC-3′ and TGL2: 5′-GCT TGT GCT TCA ATG AGC GTG TTT CG-3′). DNA/protein sequence analysis and phylogenetic analysis The sequences obtained from each strand were edited and aligned using Omiga (Oxford) software and grouped to form individual consensus sequences using SeqVerter software (GeneStudio). The nucleotide and deduced amino acid sequences were analyzed using the freely available BLAST computer programs (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences were edited and aligned using SeqVerter and BioEdit (Tom Hall, Ibis Therapeutics Division of Isis Pharmaceuticals) software following the manufacture’s manual. Multiple alignments were prepared using the ClustalX accessory in BioEdit according to Thompson et al. (1997). Neighbor-joining phylogenetic trees and maximumlikelihood trees were generated by PHYLIP 3.6.7 (http:// evolution.genetics.washington.edu/phylip.html) and drawn using Tree-View 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html). Genomic DNA/RNA extraction and RT-PCR Young tobacco leaves, which were used as the starting material for genomic DNA isolation, were extracted by CTAB. RNA from young leaves and other tissues, including root, shoot, anther, and pistil, was extracted using TRIzol reagent (Gibco BRL, New York, NY, USA). RT-PCR was performed using the SuperScript

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II Reverse Transcriptase kit (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer’s instructions. Two micrograms of total RNA were used as the template together with 1 µl oligo(dT)12~18 (25 µg µl−1). The cDNA pool was then amplified with two pairs of primers: rtl1 with rtl2, and rtu1 with rtu2 (upstream primer rtu1: 5′-GGC ATC AGC GAC TTT GAC CAA-3′; upstream primer rtu2: 5′-GCT TCC GAT TGG TTC ATC-3′; downstream primer rtl1: 5′-CTT GTT TCT TGC CAG CCT CTG-3′; downstream primer rtl2: 5′-GTG ACT TGC CCA TGG ATT-3′). Each primer combination was used for multiplex amplification with tubulin as an internal control (tbu2: 5′-CAC CAA CCT TAA CCG CCT TA-3′; tbl2: 5′-GCT GCT CAT GGT AAG CCT TC-3′; designed for Nicotiana tabacum tubA2 mRNA, NCBI Accession number AJ421412). The four nested primers (rtl1, rtl2, rtu1, and rtu2) span an intron. The predicted product lengths for the four primers varied between 180 and 250 bp, while the product length for the tbu2 and tbl2 primers was 210 bp. The PCR products were run on 2% agarose gels with a 50 bp standard (New England Biolabs, Beverly, MA, USA) or the DL2000 marker from Takara. RNAi vector construction and tobacco transformation The RNAi vector pHannibal from CSRIO can transcribe self-complementary hairpin RNA molecules aimed at dicotyledonous plants (Wesley et al., 2001). We chose the EcoRI-XhoI fragment of cDNA near the sec7 domain as an RNAi target, and the Nonconservative region between the DCB and HUS domains as another target (Fig. 1a). The constructs were named gnlHE and gnlHK, respectively. The gnlHE fragment was amplified by PCR using the primers hind3 (5′-ATC TAA GCT TTC TTG TTG GGG ATT TCT TGG-3′) and xba1 (5′-TGA TTC TAG ATT CTC GAG GGA GGT CAT TTC-3′) and inserted into pHannibal. Constructs containing two insertions were identified by PCR and sequencing. The entire RNAi components were cut by NotI digestion and transferred into the binary vector pART27. gnlHE-pART27 was transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation. Agrobacterium-mediated leaf disc transformation was performed according to the method of Horsch et al. (1985). Regenerated plants were selected on MS medium containing 100 mg l−1 kanamycin and 250 mg l−1 carbenicillin. The gnlHK-pART27 vector was constructed using the primer pairs R2XX (5′ ATA TCT AGA CTC GAG CAC TTG CTT CCG ATT GGT TC 3′) and R2HK (5′ ATA AAG CTT GGT ACC TGA CTT GCC CGT GGA TTC TA 3′). Transforming procedure was the same as that discussed earlier. Transformant validation The putative transformants were first analyzed by PCR targeted to the genomic insertion of nptII gene from the

Fig. 1 Phylogenetic tree of NtGNL1 and other GBF family members in plants. (a) Structure of NtGNL1; arrows indicate the regions targeted by RNAi. (b, c) Phylogenetic trees of NtGNL1 and other GBF family members; NtGNL1 shows closer relationship with AtGNL1 by both methods. Sources: OsGNOM, OsGNL1, OsGNL2 are from Oryza sativa (Richter et al., 2007); AtGNL1, AtGNL2, and AtGNOM are from Arabidopsis thaliana; NtGNL1 is from Nicotiana tabacum, PtGN-1, PtGN-2 and PtGNL2 are from Populus trichocarpa (gw1.XVII.958., gw1.XVII.954.1 and eugene3.00180657); OtGNOM is from Ostreococcus tauri (Ot10g01110); and CrGNOM is from Chlamydomonas reinhardtii (jgi|Chlre3|152758|).

pART27 vector, and then tested by the sense and antisense RNAi arms together with the joint neighborhood sequence of pHannibal vector, using the primers designed from sequences of gnlHE and gnlHK arms and the Hannibal vector sequence in the neighborhood of the MCS site (tes1: 5′ CAT TTC ATT TGG AGA GGA CAC G 3′; tes2: 5′ AGG CGT CTC GCA TAT CTC ATT A 3′). gnlHE lines were tested by tes2 and het1 (5′ GCC TTG AGA ATC TTT TTG GAG 3′) primers. gnlHK lines were tested by tes1 and R2HK primers or by tes1 and the truncated R2H primer (5′ GGT ACC TGA CTT GCC CGT GGA TTC TA 3′). Antibody preparation and western blotting The predicted hydrophilic region of NtGNL1 (protein sequence 433–715 amino acid residue) was cut from the T-cloned genomic fragment using NcoI and XhoI and inserted into pET30a (Novagene Co., Darmstadt, Germany). The plasmid containing E. coli. strain BL21(DE3) was induced by IPTG and then

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sonicated. Fusion proteins were purified using a Ni2+-chelating affinity chromatography column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the supplier’s protocol. Purified proteins were collected by SDS-PAGE. Protein bands stained by copper sulfate were cut from the gel for recovery. The cut gel blocks underwent dialysis twice against PBS buffer and were powdered by pestles and mixed with IFA for injection. The rabbits injected were kept at the Center for Disease Control in Hubei Province. To further validate the RNAi tobacco lines, gnlHE and gnlHK, by western blotting, total protein from the transgenic lines was extracted from 100 mg of tissue using Laemmli sample buffer. The proteins were separated by 8% SDS-PAGE; a Pierce BlueRanger pre-stained protein was included as a standard. The primary antibody was diluted 1 : 500. The secondary antibody was a horseradish peroxidase-labeled goat antibody against rabbit IgG (Amersham, 1 : 2000 dilution). Signals on the membrane were detected using the Pierce Supersignal West Pico HisProbe kit as per the manufacturer’s instructions. Seed and pollen germination Primary transgenic tobacco plants were selected on Murashige and Skoog (MS) medium supplemented with 2% sucrose (w/v), 0.1% (w/v) agar, and 0.3% (w/v) Phytagel (Sigma, St Louis, MO, USA), and 100 mg l−1 kanamycin. The plants were then transferred to soil and allowed to self-pollinate. For axenic culture, tobacco seeds were surface-sterilized for 1 min in 70% ethanol and 15 min in 10% sodium hypochlorite (4% active chlorine), and then rinsed three times with sterile water and vernalized for 48 h at 4°C. The seeds were plated on White medium (White, 1963) containing 1% sucrose to measure the germination rate. Germination frequencies were calculated after 3 wk of incubation at 25°C with 16 h of daylight in a growth chamber. At least three replicates were produced. Viability and the in vitro germination frequency of the pollen were determined as described by Touraev & Heberle-Bors (1999). Images were collected and analyzed using Image J software (http://rsb.info.nih.gov/ij/). BFA treatment of pollen germination and pollen tube growth Brefeldin A (Sigma-Aldrich, St Louis, MO, USA) were dissolved in ethanol to make stock in 10 µg ml−1 concentration. The stock was diluted by ethanol and checked by UV absorption for concentration control before use. The final concentration of BFA was 0.3 µg ml−1 for the treatment of pollen germination and 0.1 µg ml−1 for pollen tube growth. The BFA solution was added into the media at the beginning of the culture for pollen germination test or after 1 h culture for pollen tube growth test.


Embryo isolation, ovule clearing, and microscopy Embryos were isolated according to the method of Li et al. (2000). Ovule and embryo fixation and whole-mount clearing were performed according to the method of Schneitz et al. (1995). Fixed slides were observed under an inverted phase contrast microscope (Olympus ck-30) and a Leica DMIRE 2 fluorescence microscope equipped with a cooled chargecoupled device (cooled CCD camera, RS image MicroMAX Princeton Instruments, Inc., Trenton, NJ, USA). Imageprocessing and analysis were performed using MetaMorph (Universal Imaging Corporation Inc.) and Photoshop software (Adobe). Root and pollen tube measurements Control lines for the void-vector transgenic line and RNAi lines were grown in liquid White medium containing 1.0% sucrose (White, 1963). The young tobacco seedlings were spread on glass slides and either scanned or photographed. The root length and growth rate were measured and calculated using Image J. Pollen tube growth was assessed according to the method of De Graaf et al. (2005). Data analysis and image collection We measured 40–60 roots or pollen tubes in each of three to five repeated experiments; P-values were calculated using Microsoft Excel (2003). All error bars in the graphs indicate standard errors of the mean. The statistical significance of the experimental results was determined using the two-sided Student’s t-test. Cleared ovules and root tips were observed under a microscope (DMIRE 2; Leica) using differential interference contrast (DIC). Images were taken with a cooled CCD camera (microMAX; Roper Scientific-Princeton Instruments) using MetaMorph software. The root system of the seedlings was observed under a stereomicroscope (SZX12; Olympus), and images were taken with a cooled CCD camera (CoolSNAP; Roper ScientificPrinceton Instruments) using RS Image software (version 1.7.3; Roper Scientific-Princeton Instruments).

Results Gene structure and evolutionary analysis By RACE reaction we determined the whole sequence of the gene and, based on the sequence, we further structurally compared the gene with other GBF members. Our analysis characterized the gene as NtGNL1. In RACE reactions the 3′ and 5′ sequences (2777 and 2320 bp in length) of cDNA were identified individually, and the overlaps were spliced to form the full-length (4975 bp) CDS sequence of NtGNL1. A 485 bp intron was identified


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Fig. 2 Expression of NtGNL1 in various tissues of tobacco (Nicotiana tabacum). Tubulin was used as the control.

in the genomic fragment amplification, which was inserted at position 875 in the cDNA (NCBI accession number EF520731). The sequences of the NtGNL1 shows varying degrees of similarity with Arabidopsis GNOM: 945/1434 (65%) identity, 1135/1434 (79%) positives, and 18/1434 (1%) gaps. The methionine residue at position 683 in NtGNL1 corresponds to 696 M in GNOM, and the protein encoded by NtGNL1 may have the potential sensitivity to Brefeldin A (BFA). The sequence of NtGNL1 also exhibits high structural homology to Arabidopsis AtGNL1 (901/1458 (61%) identity, 1128/ 1458 (77%) positives, and 37/1458 (2%) gaps). NtGNL1 belongs to GBF ARF-GEF family; its architecture is similar to that of other large ARF-GEF family members (Mouratou et al., 2005). In particular, NtGNL1 contains the DCB, HUS, SEC7, HDS1, HDS2, and HDS3 domains present in BIG and GBF family members (Fig. 1a). We constructed a phylogenetic tree to analyze GNOM and several GNOM-like sequences (Fig. 1). In this analysis, NtGNL1 was assigned to the same class as AtGNL1, and not to that of Arabidopsis GNOM (Fig. 1b,c). This suggests that although the predicted conserved domains of NtGNL1 have high homology with those in GNOM, the gene has greater overall similarity with GNL1. According to our analysis, we designated the gene NtGNL1 (Nicotiana tabacum GNOM Like 1). NtGNL1 expression pattern Reverse transcriptase PCR showed that NtGNL1 is ubiquitously expressed in various tissues of both vegetative and reproductive organs, although the level of expression was highest in the reproductive organs, especially in the anthers (Fig. 2).

Fig. 3 Western blotting with anti-NtGNL1 to confirm the RNAi effects. (a) pHannibal expression vector; (b) expression of NtGNL1 was significantly reduced in the RNAi line gnlHE; (c) NtGNL1 expression in gnlHK lines. Anti-tubulin and anti-rubisco serum was used as an endogenous standard; wild-type SR1 was used as control.

The plants of the T1 to T2 generations were further used to confirm the effects of the RNAi construct insertion in the transgenic lines by western blotting. By this technique, NtGNL1 had an apparent molecular weight of 165 kDa, which coincides with its predicted size (Fig. S5). Western blotting confirmed that the expressions of NtGNL1 in gnlHK RNAi lines were effectively knocked down (Fig. 3c); and in gnlHE RNAi lines the expressions of NtGNL1 could even not be detected (Fig. 3b). However, the effect of RNAi was not identical among the various lines (Fig. 3c). Those lines with obvious down-regulation of NtGNL1 expression were selected for further phenotypic analysis. Inhibition of root generation of the transgenic calli

Confirmation of transgenic lines We selected 15 transgenic lines for gnlHK and 10 transgenic lines for gnlHE for phenotype analysis. Transgenic lines of both gnlHE and gnlHK were confirmed by PCR first (Supplementary material, Fig. S1). A line transformed by an empty vector and wild-type SR1 were used as the control.

Using a traditional procedure of the leaf disc method, the calli were transformed with either the empty vector or the RNAi vector under the same conditions. Shoot generation in both cases was the same as that in the wild-type. After shoots had formed on the transgenic calli, they were transferred to inductive media containing 0.1 mg l−1 IBA (3-indolebutyric acid) to



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Fig. 4 Comparison of seed germination ratio, seedling size and root growth in the transgenic and control lines of tobacco (Nicotiana tabacum). (a) Germination rate of the gnlHE lines was nearly half that of the control line (P < 0.05), (n = total number of seeds used in the analysis). Control, closed bars; gnlHK, open bars; gnlHE, hatched bars. (b) Organ size in the transgenic line gnlHE (hatched bars) compared with that in the control (closed bars); no significant difference was detected in leaf size, although the stems and roots of the transgenic plantlets were shorter (P < 0.05 and P < 0.01, respectively). (c) Root growth of line gnlHE (circles) compared with that of the control (squares); n = 18–21. (d) Root growth of line gnlHK (circles) compared with that of the control (squares); n = 35. P-values calculated for each time point were all < 0.01 except for the seventh day in (d) (P < 0.05). Error bars, ± 1SE.

induce root formation. In the controls, roots formed as well as they did in the wild-type (100%, n = 50); however, in the RNAi lines, the calli were seldom able to produce roots (1.4%, n = 69). Repeated experiments produced similar results, suggesting that the ability to generate roots was greatly inhibited in the RNAi lines. Seed germination was reduced in the RNAi transgenic lines Seed germination was assessed at the T2 generation in our RNAi lines. In the gnlHE lines analyzed, only 50.78% of the seeds germinated, which is significantly lower than in the wild-type seeds (Fig. 4a), while in the gnlHK lines analyzed, 57.01% of the seeds germinated.


To investigate why so many seeds failed to germinate, we isolated the embryos from the aborted seeds and examined their developmental stage and morphology. We found that approx. 60% of the embryos isolated were arrested at the torpedo stage (Fig. S2A), while nearly 10% of the embryos were arrested at the heart stage (Fig. S2B) in gnlHE lines. Similar phenotype was also observed in the gnlHK lines. Clearly, in some of the RNAi transgenic lines, embryo development was constrained by knocking down NtGNL1 expression. Root elongation was affected in the RNAi transgenic lines Plantlets from the gnlHE and control lines were compared in terms of their first real leaves, stems, and the length of their


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main roots. Compared with the control lines, plantlets from the gnlHE line had normal-sized leaves but significantly shorter stems. The biggest difference was observed in the roots, which were substantially shorter in the transgenic plantlets (Fig. 4b; Fig. S3). Further analysis indicated that root growth of gnlHE and gnlHK lines was not arrested at a specific stage, but slowed overall (Fig. 4c,d). In addition, the structure of the root tip, the number of stem cells, the alignment of the meristematic cell layer, and the morphology of the cells were not notably different (the date not shown) between the experimental and control plantlets (Fig. S4). Only occasionally, some root tips showed highly differentiated meristematic regions. Clearly, down-regulation of NtGNL1 expression did not seriously alter root structure or cell differentiation in the RNAi lines, although it negatively affected root elongation. Down-regulation of NtGNL1 expression resulted in altered patterns of cell division and asynchronous cellular development In wild-type plant development, the elongated zygote divides asymmetrically to produce a small apical cell and a large basal cell. The first apical cell division is transverse and forms an upper cell (UC) and a lower cell (LC). Both cells subsequently divide twice vertically and symmetrically. Both planes of division are perpendicular to each other and thus produce an eight-celled embryo proper, with four cells in the upper layer (UL) and four in the lower layer (LL); the cells share a similar morphology. Subsequently, the eight cells divide once synchronously to form a 16-celled embryo proper. This division is synchronous, and the division plane stretches around the periphery of the embryo; in this way, the radial pattern of embryo is established and the protodermal cell layer is formed (He et al., 2007). We carefully examined early embryogenesis in our RNAi transgenic plants and compared it with that in wild-type plants. In the ovules of our transgenic lines, all of the zygotes developed normally (i.e., they elongated to the right size and assumed the correct morphology; Fig. 5, panel 1). The first round of division in each transgenic zygote was transverse and asymmetric, and produced a two-celled embryo with a large basal cell and a small apical cell (Fig. 5, panel 2), similar to that in wild-type zygotes (He et al., 2007). A symmetric zygote division was never observed in the transgenic lines. In the development that followed, however, unusual patterns of division were frequently observed. As early as the two-celled embryo stage, the orientation of cell division became irregular; in both the UC and LC cells, the plane of division was diagonal or even nearly transverse (Fig. 5, panel 3). At this stage, the position of the cell division plane also became changeable, and obvious asymmetric divisions were observed that resulted in UL and LL cells with altered morphologies and volumes (Fig. 5, panels 4 and 5). Owing to the irregular division, sometimes

more than four cells were produced in the UL or LL (Fig. 5, panel 6). Unlike the situation in the wild-type, in which the eight cells in the UL and LL synchronously and peripherally divide once to produce a 16-celled embryo with a protodermal layer, in the gnlHE lines, peripheral division was found to occur in one of the cells first (Fig. 5, panel 7), only in the LL (Fig. 5, panel 8), or earlier than the second division in the UC and LC (Fig. 5, panel 9). Thus, the orientation and position of cell division were altered, and the expected sequence of embryonic cell division was interrupted in our gnlHE lines. Another notable phenotype was the asynchronous development of the cells in the embryo. Some cells grew faster than their neighbors, and therefore the typical symmetry and morphology of the embryos were modified (Fig. 5, panel 10). In some cases, as a result of differential cell expansion, cell alignment was disturbed so that the entire embryo appeared disorganized (Fig. 5, panel 11). More frequently, however, the embryos showed swelling in only some parts (Fig. 5, panel 12). These shape effects were maintained until the mature embryo stage. Compared with normal embryos at the same developmental stage (Fig. 5, panel 13), the gnlHE embryos showed various morphologies, including transversely expanded or very short hypocotyls (Fig. 5, panels 14 and 15) and unusually large and fused cotyledons (Fig. 5, panel 16). Similar phenotype was also observed in the gnlHK lines, although the frequency was much lower. However, in both the normal and abnormally shaped embryos, the apical and basal patterns were properly established, and embryonic root and cotyledon differentiation was not seriously interrupted. RNAi impeded pollen germination and pollen tube growth Anther and pollen development were examined in both gnlHE and gnlHK lines, but no notable defects were found. In addition, pollen viability, tested by FDA staining, was c. 80% on average. Unexpectedly, however, pollen germination and pollen tube growth were obviously impaired. When germinated in vitro, the control (35 s:void pHannibal-transformed line) pollen began to germinate after 0.5 h in culture medium and reached maximal germination (85%) after 2 h, whereas the pollen from some of gnlHE lines (over five individual plants from each line) had a much lower germination frequency (Fig. 6a). In extreme cases, only a few pollen tubes were able to elongate to the same extent as those in the control (Fig. 7, panels 1 and 2). Pollens from some of gnlHK lines (over five individual plants from each lines) also show significant low germination frequency (Fig. 6a). A comparison of pollen tube growth between gnlHE and the control showed that gnlHE pollen tube growth was significantly slowed, although the overall amounts of growth were equal (Fig. 6c). As a result, the average pollen tube in gnlHE was significantly shorter than that in the control.



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Fig. 5 Embryo morphology and cell division patterning in the RNAi transgenic lines (gnlHE) of tobacco (Nicotiana tabacum). 1, a wild-type zygote; 2, a wild-type two-celled embryo; 3, a four-celled embryo showing the nearly transverse and diagonal planes of division in the upper layer UL, and lower layer LL, respectively (arrows indicate the planes of division); 4, the UC and LC each divided asymmetrically and diagonally (arrows indicate the planes of division); 5, unequal division in the UL and LL resulted in altered cell volumes (arrows indicate the plane of division); 6, irregular cell division produced more cells in the LL arrows than in the UL; 7, asynchronous embryonic cell division (arrow indicates peripheral cell division in one cell); 8, peripheral cell division occurred earlier in the LL (arrows indicate the planes of cell division); 9, when peripheral division was complete, the LC remained undivided; 10, owing to asynchronous development, the typical symmetry of the embryo was altered (the arrow indicates the expanded cell); 11, a disorganized 16-celled embryo; 12, a small globular embryo showing swelling in the LL derived part arrows; 13, a nearly mature wild-type embryo; 14, an embryo with a swelled hypocotyl and protruding tissue arrow, that looked like the primordium of a third cotyledon; 15, a mature embryo with a very short hypocotyl; 16, an embryo with expanded and fused cotyledons. Bar, 5 µm (1, 2); 10 µm (3–8); 20 µm (9–12); 40 µm (13–16).

The effect of BFA treatment on pollen germination and pollen tube growth According to its 683M NtGNL1 has similar character with those predicted BFA-sensitive GBF members (Geldner et al.,


2003). We tested the effect of BFA treatment on both pollen germination and pollen tube growth. When incubated in the medium with 0.3 µg ml−1 BFA, the wild-type pollen germination was obviously inhibited (Fig. 6b). As illustrated in the same figure, BFA treatment has dosage effect. Lower


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than 0.05 µg ml−1 BFA in the medium pollen germination was almost normal, but at higher than 0.5 µg ml−1 BFA, pollen germination could be totally inhibited (data not shown here). The inhibition effect in BFA treatment was much stronger than that of RNAi and this was further confirmed by the fact that pollen germination ratio of RNAi transgenic lines was further lowered after BFA treatment (Fig. 6b). The treatment of BFA in very low concentrations also obviously inhibited pollen tube growth (Fig. 6c), but low-concentration BFA (0.05–0.1 µg ml−1) did not significantly restrain pollen tube growth of RNAi transgenic lines (data not shown here). Morphological variation of pollen tubes in transgenic lines Morphological observation revealed various abnormalities in the pollen tubes of the gnlHE lines. Most notable was the formation of helical pollen tubes (Fig. 7, panel 3). The pollen tubes appeared to change their direction of growth frequently during elongation; as a result, branched (Fig. 7, panel 4), zigzagged (Fig. 7, panel 5), bulging (Fig. 7, panel 6), or bubbled (Fig. 7, panel 7) pollen tubes were frequently observed at the start of germination. Although the pollen tubes varied in morphology, sperm cell formation appeared normal (Fig. 7, panels 8 and 9). We further analyzed pollen tube growth in vivo by employing an in vivo–in vitro technique (Sun et al., 2000), which confirmed that the pollen of the gnlHE line grew more slowly than that of the control. This was clearly indicated by the fact that the control pollen tubes appeared earlier than those of the gnlHE lines, and they were much longer (Fig. 7, panels 10 and 11).

Discussion NtGNL1 may be involved in maintaining the normal cell division pattern during embryogenesis

Fig. 6 Pollen germination and pollen tube growth were restrained by RNAi and brefeldin A (BFA) treatment in tobacco (Nicotiana tabacum). (a) Comparison of pollen germination in gnlHE (hatched bars), gnlHK (open bars) and the control (closed bars) in vitro. (b) Pollen germination ratio of gnlHE lines was further reduced after BFA treatment. BFA final concentration was 0.3 µg ml−1 in pollen culture medium and data shown here were collected after 3 h BFA treatment. Treatments: free of BFA, closed bars; 0.2 µg ml−1 BFA, open bars; 0.3 µg ml−1 BFA, hatched bars). (c) Pollen tube growth in gnlHE (squares) transgenic plants compared with the control (circles) and BFA (0.065 µg ml−1; triangles) treated pollen tubes. P < 0.01 for each time point (control vs gnlHE or control vs wild-type + BFA). Error bars, ± 1SE.

The large ARF-GEF family comprises two subfamilies, BIG and GBF (also called the GBG family). BIG includes human BIG1 and BIG2, and yeast Sec7p, while GBF consists of human GBF1, Arabidopsis GNOM, and yeast Gea1 and Gea2. All members of this family are thought to be involved in sorting proteins and other materials during vesicular trafficking from the endo-membrane system; most have been shown to perform their functions at the Golgi body, and only BIG2 and GNOM have been reported as localized to endosomes (Jürgens & Geldner, 2002). Five BIGs (BIG1–5) and three GBFs (Gnom, GNL1, and GNL2) have been found in Arabidopsis (Jürgens & Geldner, 2002). Additional BIGs/GBFs were recently discovered in rice and other species. Insertion mutations in the BIG family member At1g01960 result in variable numbers of nuclei or a lack of embryo sac formation (Pagnussat et al., 2005). Recently published works further confirmed that GNOM and GNL1



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Fig. 7 Comparison of pollen tube growth of tobacco (Nicotiana tabacum) in vitro and in vivo. 1, gnlHE pollen germination, showing few germinated pollen grains; 2, germinated control pollen; 3, pollen tubes from the gnlHE lines showing a helical morphology arrows; 4–7, abnormalities in pollen tube morphology in the gnlHE lines: branching (4, arrow), zigzagging (5), tip expansion (6), and bubbling (7); 8, 9, a pair of sperm cells in a pollen tube shown by fluorescence microscopy using DAPI-labeled sperm under a weak bright-field (8), and a fluorescent image of the same sperm cells under a dark-field (9); 10, 11, comparison of pollen tube growth in vivo in the transgenic (10) and wild-type (11) lines. The photographs were taken 48 h after pollination. Bar, 100 µm (1, 2); 50 µm (3); 20 µm (4–7); 5 µm (8).

share a common, essential function in ER-Golgi trafficking, whereas only GNOM has another, unique essential function in endosomal recycling (Richter et al., 2007). The accumulated data reveal that GNOM plays a critical role in apical-basal axis formation during early embryogenesis. Strong gnom mutants produce zygotes with symmetric division, and as a result, the patterning of the embryo body is severely altered. Thus, the cell files in those embryos are disorganized with disrupted apical-basal patterning (Mayer et al., 1993). Careful analysis of weak gnom alleles has confirmed the critical role of this gene in maintaining primary root meristematic activity and in the initiation of lateral root primordia by mediating auxin transport. The typical phenotype of weak gnom mutants includes roots that eventually cease to grow because of the collapse of the root meristem, and which differentiate into mature tissues (Geldner et al., 2004). Phenotypic analysis of our RNAi transgenic lines indicated that embryonic cell division was variable and resulted in embryos with various abnormal structures and body shapes. Seed germination was also greatly reduced, and root growth was significantly inhibited. These results suggest that NtGNL1 is involved in embryogenesis and postembryonic development in ways that are distinct from those of gnom. Firstly, down-


regulation of NtGNL1 produced variable planes of cell division during early embryogenesis. While this disturbed cell alignment within the embryos, radial and apical-basal patterning was not markedly disturbed. Therefore, although root initiation was inhibited and other abnormalities, such as fused cotyledons, very short hypocotyls, and expanded radial structures, were observed, the developmental axis was established correctly. Second, the RNAi transgenic line exhibited asynchronous cell division and growth during early embryogenesis. Since the sequence of cell division was disturbed, the symmetry and shape of the embryos were modified; in particular, some of the cells underwent rapid division and growth whereas other cells were delayed. NtGNL1 may also function in later embryo and postembryonic development. Nearly half of the embryos were arrested at the heart or torpedo stages, and root elongation in seedlings of the transgenic line was significantly slower than that in the control. Unlike those in the gnom mutant, the root tips in the lines were fully differentiated and active. Lateral root generation was also normal. This indicates that the gene may not function in maintaining meristem cells, but is likely involved in maintaining an appropriate pace of embryonic root development.


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NtGNL1 is also involved in pollen germination and pollen tube growth In gnom mutants, pollen tube growth is not different from that in wild-type plants. GUS staining has shown that GNOM of Arabidopsis is not expressed in anthers, and only weakly in pollen grains (Geldner et al., 2004). However, in some of our RNAi lines, pollen germination was severely inhibited even though pollen viability was similar to that in the wild-type. Pollen tube growth in these RNAi transgenic lines was also much slower than that in the control. These results were further confirmed by an in vitro–in vivo technique, which showed that both in vitro and in vivo pollen tube growth was inhibited in the transgenic lines. Pollen tube growth is a polarized growth process whereby the tips of the growing tubes elongate to deliver sperm cells for fertilization (De Graaf et al., 2005). During this process, vesicle trafficking is essential for establishing and maintaining the polarity of pollen tubes (Samaj et al., 2006). This is one of the most important tip-growth models in higher plants, and it is regulated by membrane-associated small GTPases (Samaj et al., 2006). Tip growth in pollen tubes is thought to be regulated by exocytosis and endocytosis around the apical dome. If endosomal transportation is blocked, pollen-tube tip growth should be affected. However, among the investigated members of the GBF family that contain the sec7 domain and are predicted to function in vesicle trafficking, none has been confirmed to be involved in regulating pollen tube growth. According to Richter et al., (2007), gnl1 pollen competition in Arabidopsis was reduced to 70%. Although the details of male gametophyte development in gnl1 are unknown, this result also suggests that GNL1 is involved in male transmission, which is consistent with the results reported here. It was reported that BFA could block vesicular secretion in tobacco pollen tubes, thereby blocking the deposition of cell wall materials and resulting in arrested pollen tube growth (Parton et al., 2003). Furthermore, in Picea meyeri, BFA inhibited pollen germination and pollen tube growth (Wang et al., 2005). These results further confirm that vesicular secretion and trafficking are critical for pollen tube germination and growth. Knocking down the genes that maintain normal vesicular trafficking should inhibit pollen tube germination and growth. However, the targets of BFA in pollen tubes have not yet been identified. The functions of the GBF family members in regulating pollen tube growth are largely unknown. Brefeldin A can inhibit the activation of small GTP-binding proteins by stabilizing an abortive complex between a G-protein and the catalytic sec7 domain found in some GEFs. The binding site of BFA in the sec7 domain has been identified (Renault et al., 2003). Geldner et al. (2003) found that in the GBF-BIG family, a specific amino acid residue in a conservative position of the sec7 domain (e.g. the 696M amino acid in GNOM) reflects the whole protein’s sensitivity to BFA in most cases: methionine may be sensitive to BFA, and leucine

may be resistant to BFA. Geldner et al. (2003) used genetic engineering methods to replace the 696M with Leu, after which the protein became resistant to BFA. Based on experimental evidence, the GBF-BIG family members can be broadly divided into BFA-sensitive and BFA-resistant. GNL1 of Arabidopsis was predicted to be BFA-resistant since it has Leu, not Met, in that position. However, we found that NtGNL1 has the proper 683M residue in the conservative position within the sec7 domain. We further treated pollen and pollen tubes of wild-type tobacco with BFA and found that both pollen germination and pollen tube growth were inhibited. This is similar to the phenotype in NtGNL1 RNAi transgenic plants. Thus, NtGNL1 might be one of BFA targets in pollen tubes. Ritzenthaler et al. (2002) treated tobacco BY-2 cells with BFA and found a rapid effect of BFA on Golgi morphology, suggesting that the tobacco GNL1 ortholog is BFA-sensitive. Therefore, we propose that the inhibition of pollen germination and tube growth in NtGNL1 RNAi transgenic plants might also result from the disruption of vesicular trafficking. Comparison of gnlHE and gnlHK phenotypes to distinguish the function of NtGNL1 Under extreme conditions, off-target effects in RNAi transgenic plants might knock down homologous genes that have only 11 continuous identical nucleic acid sequences, although at a very low frequency (Jackson et al., 2003). Because the gnlHE fragment is near the sec7 conservative domain, its nucleic acid sequence shows over 70% homology to both GNOM and GNL1 at some sites in Arabidopsis. To estimate the possibility of knocking down both homologous genes in gnlHE lines, we chose another gnlHK fragment that is not in the conservative domain and made corresponding RNAi transgenic lines for comparison. Sequence analysis indicated that the gnlHK region shares less than 50% homology with GNOM and GNL1 of Arabidopsis and even less homology with other genes. The fragment we chose rarely has over 15 continuous nucleic acid sequences identical to the NtGNL1 homolog. Thus, it is a useful control in phenotypic analysis of RNAi transgenic lines for distinguishing the function of NtGNL1. Comparisons between the phenotypes of gnlHE and gnlHK transgenic lines show that they share most phenotypes. In both lines, a low seed germination ratio and inhibited root growth were observed, although certain gnlHK transgenic lines had root growth similar to that of wild-type plants. During embryogenesis, irregular embryonic cell division and development were observed in both lines. The pollen germination ratio was also reduced in gnlHK lines. Generally, the abnormalities in gnlHE lines were more severe than those in gnlHK lines. In particular, pollen tube morphology in all gnlHK lines was generally normal, although pollen germination and pollen tube growth were inhibited in some gnlHK lines. A similar phenomenon was observed in BFA-treated pollen tubes. This suggests that, in gnlHE lines, expression of genes other than



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NtGNL1 was possibly affected. According to Richter et al. (2007), the double mutant of gnl1 gnom is lethal, whereas a single gene mutant of gnl1-2 revealed multiple abnormalities in root and ovule development (Teh & Moore, 2007). Thus, although the expression of both NtGNOM and NtGNL1 may be affected in gnlHE transgenic lines, the down-regulation of NtGNL1 is likely responsible for the main phenotypes. Together with the data from the gnlHK transgenic lines, our analysis indicates that NtGNL1 should be involved, although perhaps not uniquely, in both vegetative organ growth and sexual reproduction in plants.

Acknowledgements The project was supported by the ‘973’ Project (2007CB108700), National Natural Science Fund of China (90408002, 30521004) and the 111 Project. We thank Prof. Jürgens G. in Tubingen University for his useful suggestions. We also thank Dr Jingzhe Guo and Biao Wang for technical assistance in preparation of the antibody and western blot. We thank CSRIO for kindly sending us the pHannibal vectors.

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Supplementary Material The following supplementary material is available for this article online: Fig. S1 Confirmation of our RNAi transgenic lines by PCR. Fig. S2 Arrested embryos in ungerminated seeds. Fig. S3 Root growth in the wild-type control and in a transgenic line.

Fig. S4 Root tip structure in the wild-type control and in an RNAi transgenic line. Fig. S5 Western blotting showing that the antibody against NtGNL1 was efficient and protein products were at the approximate molecular weight 165 KD. This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/ 10.1111/j.1469-8137.2008.02444.x (This link will take you to the article abstract). Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the journal at New Phytologist Central Office.

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