TY - JOUR
AU - Wang, Co-Shine
AB - Abstract Here, we report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) gene is regulated during the stage of pollen maturation. A gene encoding Rho GTPase was identified in lily (Lilium longiflorum Thunb.) pollen. Phylogenetic tree analysis of lily LLP-Rop1 revealed that the protein shares greatest similarity with Group 4 Rops. The LLP-Rop1 gene was spatially and temporally regulated in lily plants during anther development. Accumulation of the LLP-Rop1 transcript decreased its level of accumulation while LLP-12-2, a Rop-interactive CRIB motif-containing (RIC) transcript increased either by premature drying of developing anther/pollen or by the exogenous application of various concentrations of abscisic acid (ABA) during pollen maturation and tube growth. Application of norflurazon, an ABA biosynthesis inhibitor, also resulted in the downregulation of the LLP-Rop1 gene while LLP-12-2 was upregulated by ABA. Furthermore, an increase in ABA in the maturing pollen correlated with desiccation that occurred in the anther prior to anthesis. LLP-Rop1 overexpression inhibited tube elongation, and caused tube expansion and the formation of a ballooned tip. CFP–LLP-Rop1 was localized to the cytoplasm having a greater intensity along the tube plasma membrane. Fluorescence resonance energy transfer analysis of lily pollen tubes coexpressing CFP–LLP-Rop1 and YFP–LLP-12-2 demonstrated that LLP-12-2 is a target RIC protein of active LLP-Rop1, but the interaction between LLP-Rop1 and LLP-12-2 proteins is probably irrelevant of dehydration in the dried pollen. Introduction Pollen development is a sophisticated differentiation process. The process of pollen maturation is controlled by a large pool of ‘late genes’ (McCormick 1993). When pollen grows to full maturity it exhibits various degrees of desiccation. The desiccation process prior to anthesis is accompanied by expression of a set of late genes. Although changes in expression of individual late genes during pollen development have been described in various plant species (Scholz-Starke et al. 2003, Filichkin et al. 2004, Miki-Hirosige et al. 2004), limited information exists about the genes related to desiccation. A few pollen gene products related to desiccation have been previously reported (Detchepare et al. 1989, Bedinger and Edgerton 1990). Recently, >30 genes were identified from a cDNA library constructed at the desiccation period of anther development (Hsu et al. 2007). LLP-Rop1 is one of those genes associated with desiccation. Moreover, we previously identified LLP-12-2, a gene encoding Rho GTPase of plants (Rop)-interactive Cdc42/Rac-interactive binding (CRIB) motif-containing protein (RIC) that is also dehydration inducible (Hsu and Wang 2006). It is known that RICs are target proteins of Rop GTPases (Wu et al. 2001). Rop GTPases are a family of plant-specific signaling molecules solely representing the Ras/Rho family of Ras-related G proteins in plants (Nibau et al. 2006). Numerous studies have shown that Rop GTPases play major roles in polarity determination in tip-growing tubes (Yang and Fu 2007, Kost 2008). The Rop-mediated effect on polar growth is achieved via regulation of multiple cellular response pathways that involve the formation of a tip-focused cytoplasmic Ca2+ gradient, the reorganization of actin cytoskeleton (Gu et al. 2005) and the production of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] at the pollen tube tip membrane (Kost et al. 1999). Moreover, different RICs may act as Rop targets to control different Rop-dependent pathways in pollen tubes (Gu et al. 2005). Studies have revealed multiple functional roles for Rops in plant growth and development, acting as key regulators not only for polarity establishment in cell growth and differentiation, but also for signaling in disease and abiotic stresses (Gu et al. 2004, Nibau et al. 2006). It was reported that rice OsRacB is associated with salt tolerance (Luo et al. 2006). Arabidopsis Rop2 may activate the levels of reactive oxygen species in both the whole leaf and single cells and Rop 10 GTPase is involved specifically in the negative regulation of abscisic acid (ABA) signaling (Zheng et al. 2002, Park et al. 2004). Nevertheless, no information about Rops and its interacting proteins with response to desiccation have been reported to date. We report desiccation-associated ABA signaling transduction through which the LLP-Rop1 gene is regulated during the stage of pollen maturation. The LLP-Rop1 and LLP-12-2 genes are expressed differently in response to both desiccation and other developmental cues at the pollen maturation phase during anther development. Both genes are upregulated by other developmental cues but upon desiccation, the LLP-Rop1 and LLP-12-2 genes are antagonistically responsive. We demonstrate that the LLP-Rop1 protein interacts with LLP-12-2 in vivo using fluorescence resonance energy transfer (FRET) analysis but dehydration probably does not affect the interaction between LLP-Rop1 and LLP-12-2 proteins in dried pollen although it affects the expression of the LLP-Rop1 and LLP-12-2 genes. Results LLP-Rop1 shares greatest similarity with Group 4 Rop GTPases LLP-Rop1 cDNA (accession number EF140700) was identified from an enriched cDNA library constructed from anthers at the desiccation stage of pollen maturation (Hsu et al. 2007). The LLP-Rop1 gene contains an open reading frame of 594 bp encoding a polypeptide of 197 amino acids with a calculated molecular mass of 21.7 kDa and a pI of 9.1. Sequence analysis revealed significant similarity between the predicted LLP-Rop1 and other Rop GTPases (Fig. 1). LLP-Rop1 possesses a fragment of conserved effector domain from 29 to 49 amino acid residues, similar to other Rops. In addition, the protein has the combination of a CXXL motif at the end of the C-terminus with a polybasic domain of six lysine residues at the proximal site of the CXXL motif both required for plasma membrane targeting (Hancock et al. 1991). LLP-Rop1 has >90% amino acid residues identical to rice OsRacD (accession no. Q6EP31), barley HvRacB (accession no. CAC83043) and Arabidopsis AtRop1 (accession no. P92978), but a much lower percentage of common identity with rice OsRac3 (74%, accession no. Q6Z808), maize Rop6 (71%, accession no. CAB96793) and Arabidopsis AtRop7 (81%, accession no. Q38903). Phylogenetic tree analysis indicates that known Rops are classified into four groups among which LLP-Rop1 shares the highest similarity with members of Group 4 (Fig. 2). Fig. 1 View largeDownload slide Alignment of lily LLP-Rop1 with related Rops. The lily LLP-Rop1 amino acid sequence is compared with the predicted amino acid sequences of related Rops: rice OsRacD (accession no. Q6EP31), barley HvRacB (accession no. CAC83043) and Arabidopsis AtRop1 (accession no. P92978) in Group 4; rice OsRac3 (accession no. Q6Z808) in Group 1; maize ZmRop6 (accession no. CAB96793) in Group 2 and Arabidopsis AtRop7 (accession no. Q38903) in Group 3. A dash in the sequence indicates a gap introduced in order to maintain good alignment. The box indicates the conserved effector domain. Underline indicates the CXXL motif. Amino acids that are identical to lily LLP-Rop1 are marked with black boxes. The Multiple Sequence Alignment application (AlignX) of the Vector NTI Suite program (InforMax, Inc., North Bethesda, MD, USA), based on the Clustal W algorithm, was used. Fig. 1 View largeDownload slide Alignment of lily LLP-Rop1 with related Rops. The lily LLP-Rop1 amino acid sequence is compared with the predicted amino acid sequences of related Rops: rice OsRacD (accession no. Q6EP31), barley HvRacB (accession no. CAC83043) and Arabidopsis AtRop1 (accession no. P92978) in Group 4; rice OsRac3 (accession no. Q6Z808) in Group 1; maize ZmRop6 (accession no. CAB96793) in Group 2 and Arabidopsis AtRop7 (accession no. Q38903) in Group 3. A dash in the sequence indicates a gap introduced in order to maintain good alignment. The box indicates the conserved effector domain. Underline indicates the CXXL motif. Amino acids that are identical to lily LLP-Rop1 are marked with black boxes. The Multiple Sequence Alignment application (AlignX) of the Vector NTI Suite program (InforMax, Inc., North Bethesda, MD, USA), based on the Clustal W algorithm, was used. Fig. 2 View largeDownload slide Phylogenetic tree of Rop genes. Phylogenetic tree produced after the analysis of 40 publicly available plant Rho family nucleotide sequences including all accessible Rop genes of Arabidopsis, rice and maize. At, Arabidopsis (Arabidopsis thaliana); Br, turnip (Brassica rapa); Ca, chickpea (Cicer arietinum); Gh, upland cotton (Gossypium hirsutum); Gm, soybean (Glycine max); Hv, barley (Hordeum vulgare); Lj, Lotus japonicus; Mt, barrel medic (Medicago truncatula); Nt, tobacco (Nicotiana tabacum); Os, rice (Oryza sativa); Pp, moss (Physcomitrella patens); Tv, spiderwort (Tradescantia virginiana); Ze, zinnia (Zinnia elegans); Zm, maize (Zea mays). Fig. 2 View largeDownload slide Phylogenetic tree of Rop genes. Phylogenetic tree produced after the analysis of 40 publicly available plant Rho family nucleotide sequences including all accessible Rop genes of Arabidopsis, rice and maize. At, Arabidopsis (Arabidopsis thaliana); Br, turnip (Brassica rapa); Ca, chickpea (Cicer arietinum); Gh, upland cotton (Gossypium hirsutum); Gm, soybean (Glycine max); Hv, barley (Hordeum vulgare); Lj, Lotus japonicus; Mt, barrel medic (Medicago truncatula); Nt, tobacco (Nicotiana tabacum); Os, rice (Oryza sativa); Pp, moss (Physcomitrella patens); Tv, spiderwort (Tradescantia virginiana); Ze, zinnia (Zinnia elegans); Zm, maize (Zea mays). Spatial and temporal accumulation of LLP-Rop1 transcripts during pollen development To determine the spatial distribution of LLP-Rop1 in lily plants, reverse transcriptase (RT)–PCR analysis was used. Gene-specific primers were designed according to sequences of the 5′- and 3′-untranslated regions for the gene (Table 1). As shown in Fig. 3A, the LLP-Rop1 transcript was detected not only in the anther but also in the filament, stem and root. It is worthy of note that LLP-12-2, an effector of LLP-Rop1, is pollen specific (Hsu and Wang 2006). Table 1 Primer sequences used in the work Primer  Sequence  rRNA-f  5′-GGACAGTCGGGGGCATTCGTAT-3′  rRNA-r  5′-CCAGACAAATCGCTCCACCAAC-3′  LLP-Rop1-f  5′-GGCTTCCTTTCTCTGTCTTC-3′  LLP-Rop1-r  5′-CACATACAAACCACATTCTCG-3′  CA-llp-rop1-f  5′-pTACCGCAGGTCTGGAGGACTAC-3′  CA-llp-rop1-r  5′-pTCCCACAAACCAAGGTTTACCG-3′  DN-llp-rop1-f  5′-pGGA CGA AAC TCG CTC TCC GTG ATG-3′  DN-llp-rop1-r  5′-pCAACAAGAATTATAGGCACTCCGGGAG-3′  EcoRI–LLP-Rop1-f  5′-GAATTCCGGATGAGCGCGTCGAGGTTC-3′  LLP-Rop1–SacI-r  5′-CGAGCTCGTCACAAAATGGAGCACCC-3′  LLP-Rop1–FL-5′  5′-ATGAGCGCGTCGAGGTTC-3′  LLP-Rop1–FL-3′  5′-TCACAAAATGGAGCACCC-3′  BamHI–CFP- f  5′-CGGGATCCCGATGGTGAGCAAGGGCGA GGAG-3′  CFP–EcoRI-r  5′-CCGGAATTCCTTGTACAGCTCGTCCATGCC-3′  CFP–SacI-r  5′-CGAGCTCGTTACTTGTACAGCTCGTC-3′  EcoRI–LLP-12-2-f  5′-CCGGAATTCATGGGGACCAAGATGAAG-3′  LLP-12-2–SacI-r  5′-CGAGCTCGTCACACAAGACACCCCTC-3′  LLP-12-2–T3-g1  5′-ATGGGGGACCAAGATGAAAG-3′  LLP-12-2–T7-g1  5′-CACAAGACACCCCTCCTC-3′  LLA-23-5′-over-1  5′-ATGGCCGAGGAGCACCACAA-3′  LLA-23-3′-over-1  5′-ACCGAAGAAGTGGTGCTTCT-3′  Primer  Sequence  rRNA-f  5′-GGACAGTCGGGGGCATTCGTAT-3′  rRNA-r  5′-CCAGACAAATCGCTCCACCAAC-3′  LLP-Rop1-f  5′-GGCTTCCTTTCTCTGTCTTC-3′  LLP-Rop1-r  5′-CACATACAAACCACATTCTCG-3′  CA-llp-rop1-f  5′-pTACCGCAGGTCTGGAGGACTAC-3′  CA-llp-rop1-r  5′-pTCCCACAAACCAAGGTTTACCG-3′  DN-llp-rop1-f  5′-pGGA CGA AAC TCG CTC TCC GTG ATG-3′  DN-llp-rop1-r  5′-pCAACAAGAATTATAGGCACTCCGGGAG-3′  EcoRI–LLP-Rop1-f  5′-GAATTCCGGATGAGCGCGTCGAGGTTC-3′  LLP-Rop1–SacI-r  5′-CGAGCTCGTCACAAAATGGAGCACCC-3′  LLP-Rop1–FL-5′  5′-ATGAGCGCGTCGAGGTTC-3′  LLP-Rop1–FL-3′  5′-TCACAAAATGGAGCACCC-3′  BamHI–CFP- f  5′-CGGGATCCCGATGGTGAGCAAGGGCGA GGAG-3′  CFP–EcoRI-r  5′-CCGGAATTCCTTGTACAGCTCGTCCATGCC-3′  CFP–SacI-r  5′-CGAGCTCGTTACTTGTACAGCTCGTC-3′  EcoRI–LLP-12-2-f  5′-CCGGAATTCATGGGGACCAAGATGAAG-3′  LLP-12-2–SacI-r  5′-CGAGCTCGTCACACAAGACACCCCTC-3′  LLP-12-2–T3-g1  5′-ATGGGGGACCAAGATGAAAG-3′  LLP-12-2–T7-g1  5′-CACAAGACACCCCTCCTC-3′  LLA-23-5′-over-1  5′-ATGGCCGAGGAGCACCACAA-3′  LLA-23-3′-over-1  5′-ACCGAAGAAGTGGTGCTTCT-3′  BamHI, EcoRI and SacI sites are marked with bold letters. View Large Fig. 3 View largeDownload slide Spatial and temporal expression of the LLP-Rop1 gene in L. longiflorum. (A) RT–PCR was performed on total RNA isolated from vegetative organs: root, stem and leaf, and from parts of floral organs: tepal, anther, filament, stigma, style and ovary of >15-cm buds. (B) RT–PCR was performed on total RNA isolated from anthers of different size classes of buds: 1, <1.5-cm buds; 2, 2.5-cm buds; 3, 4.5-cm buds; 4, 6.5-cm buds; 5, 8-cm buds; 6, 10-cm buds; 7, 13-cm buds; 8, >15-cm buds. The sequence of LLP-Rop1 was amplified. The 18S ribosomal gene was used as an internal control (bottom). Fig. 3 View largeDownload slide Spatial and temporal expression of the LLP-Rop1 gene in L. longiflorum. (A) RT–PCR was performed on total RNA isolated from vegetative organs: root, stem and leaf, and from parts of floral organs: tepal, anther, filament, stigma, style and ovary of >15-cm buds. (B) RT–PCR was performed on total RNA isolated from anthers of different size classes of buds: 1, <1.5-cm buds; 2, 2.5-cm buds; 3, 4.5-cm buds; 4, 6.5-cm buds; 5, 8-cm buds; 6, 10-cm buds; 7, 13-cm buds; 8, >15-cm buds. The sequence of LLP-Rop1 was amplified. The 18S ribosomal gene was used as an internal control (bottom). To assess the expression pattern of LLP-Rop1 during anther development, the transcript level of LLP-Rop1 at various stages of anther/pollen development was analyzed using RT –PCR. As shown in Fig. 3B, the LLP-Rop1 transcript was first detected in the developing anther of 6.5-cm buds where microspore mitosis occurred. Afterwards, the LLP-Rop1 transcript accumulated to a maximum level in the anther prior to anthesis (15.5- to 16.5-cm buds). The transcript was detected neither in the premeiotic phase nor in the microspore development phase. Effect of premature drying and exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts during anther/pollen development Since LLP-12-2 (accession number AF477624) encoding a RIC protein was previously identified (Hsu and Wang 2006), expression of LLP-12-2 along with LLP-Rop1 gene during anther/pollen development was examined by quantitative RT–PCR (Q-PCR) and RNA blot analysis. To determine the drying effect on the accumulation of both transcripts, premature anthers of indicated bud lengths were removed and air-dried for 30 h. The water content in the anther of indicated bud lengths dropped from 63% to 14% of the fresh weight after drying. Total RNA was then extracted from developing anthers from various bud sizes. Q-PCR analysis indicated that the LLP-Rop1 transcript decreased its level of accumulation upon premature drying whereas the LLP-12-2 transcript increased (Fig. 4A). RNA blot analysis revealed similarly that the LLP-Rop1 transcript decreased its level of accumulation whereas the LLP-12-2 transcript increased upon premature drying (Fig. 4A). Lily LLA-23, an identified desiccation-related gene (Huang et al. 2000), was used as a positive control. Fig. 4 View largeDownload slide Effect of premature drying and exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in developing anthers of L. longiflorum. (A) Fresh anthers (control) were removed from indicated bud sizes and half of them were air-dried on the laboratory bench for 30 h at 25°C. (B) Freshly cut lily plants with indicated buds were dipped in aqueous solutions without (control) or with the addition of 10 or 100 μM ABA for 24 h. Total RNA was isolated from anthers of indicated bud sizes. The RNA levels of LLP-Rop1 and LLP-12-2 were determined by Q-PCR (upper panel) and RNA blot hybridization (lower panel). For Q-PCR, the data were obtained from three independent experiments. Error bars represent SD. For RNA blot analysis, RNA samples (20 μg) were denatured, fractionated on formaldehyde–agarose gels, transferred onto membranes and hybridized to the 32P-labeled cDNA isolated from corresponding plasmids. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel (bottom). Fig. 4 View largeDownload slide Effect of premature drying and exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in developing anthers of L. longiflorum. (A) Fresh anthers (control) were removed from indicated bud sizes and half of them were air-dried on the laboratory bench for 30 h at 25°C. (B) Freshly cut lily plants with indicated buds were dipped in aqueous solutions without (control) or with the addition of 10 or 100 μM ABA for 24 h. Total RNA was isolated from anthers of indicated bud sizes. The RNA levels of LLP-Rop1 and LLP-12-2 were determined by Q-PCR (upper panel) and RNA blot hybridization (lower panel). For Q-PCR, the data were obtained from three independent experiments. Error bars represent SD. For RNA blot analysis, RNA samples (20 μg) were denatured, fractionated on formaldehyde–agarose gels, transferred onto membranes and hybridized to the 32P-labeled cDNA isolated from corresponding plasmids. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel (bottom). It is expected that ABA, the level of which increases under dehydration conditions, plays a role in the regulation of gene expression. With application of either 10 or 100 μM ABA, the two genes responded differently to ABA in young anthers; the LLP-Rop1 gene was downregulated whereas the LLP-12-2 gene was upregulated when samples were analyzed by both Q-PCR and RNA blot hybridization (Fig. 4B). The ABA-inducible gene LLA-23 was used as a positive control (Huang et al. 2000). This result suggests that expression of the two genes in the anther is regulated via the ABA signaling pathway. Effect of exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts during pollen germination In order to parallel the investigation of the responsive effect of ABA on the two genes, an assay of pollen germination in vitro was used. This was possible by taking advantage of the possible disappearance of mRNA during pollen germination (Fig. 5). After incubation with various concentrations of ABA in germination buffer for various time intervals, total RNA was extracted from pollen/pollen tubes. RT-PCR analysis revealed that with application of various concentrations of ABA, the LLP-Rop1 transcript significantly decreased its level of accumulation compared with the samples without ABA addition (Fig. 5). In contrast, the LLP-12-2 transcript increased its level of accumulation in the germinating tubes when ABA was applied. The LLA-23 gene induced by ABA was used as a positive control. This result is consistent with the previous assay where exogenous ABA was applied to the young anthers (Fig. 4B). It should be noted that a substantial amount of transcript from the two genes remained in the germinating pollen even after 24-h incubation, suggesting that the two genes must play a functional role during tube growth (Fig. 5). Fig. 5 View largeDownload slide Effect of exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in L. longiflorum pollen during germination. Pollen harvested 1 d after anthesis was incubated in a germination buffer at 30°C for various time intervals. RT–PCR was performed on total RNA isolated from ungerminated and germinated pollen. The sequences of LLP-Rop1 and LLP-12-2 were amplified. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. The 18S ribosomal gene was used as an internal control (bottom). Fig. 5 View largeDownload slide Effect of exogenous ABA on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in L. longiflorum pollen during germination. Pollen harvested 1 d after anthesis was incubated in a germination buffer at 30°C for various time intervals. RT–PCR was performed on total RNA isolated from ungerminated and germinated pollen. The sequences of LLP-Rop1 and LLP-12-2 were amplified. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. The 18S ribosomal gene was used as an internal control (bottom). Effect of norflurazon on the accumulation of LLP-Rop1 and LLP-12-2 transcripts during anther/pollen maturation To further confirm that the two genes were actually affected by way of ABA signaling, norflurazon, an ABA biosynthesis inhibitor was used. As indicated in an earlier report (Wang et al. 1996), desiccation in the anther occurs about 1 d prior to anthesis. Total RNA was extracted from anthers of 9.5-cm lily buds treated without (control) or with various concentrations of norflurazon and grown to reach anthesis (15.5- to 16.5-cm buds). As shown in Fig. 6, the transcripts of LLP-Rop1 and LLP-12-2 increased their levels of accumulation as anthers grew to maturity. With the application of various concentrations of norflurazon, the LLP-12-2 mRNA decreased while LLP-Rop1 mRNA maintained its level of accumulation. RNA blot analysis revealed that treatment with norflurazon caused the known ABA-inducible LLA-23 transcript to decrease in comparison with that without treatment. Fig. 6 View largeDownload slide Effect of norflurazon on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in developing anthers of L. longiflorum. Freshly cut lily plants with buds of 9.5 cm were dipped in an aqueous solution without or with the addition of 10 or 100 μM norflurazon and grown to reach anthesis (15.5-cm buds). Total RNA was isolated from anthers of various bud sizes. RNA levels of LLP-Rop1 and LLP-12-2 were determined by Q-PCR and RNA blot hybridization. For Q-PCR, the data were obtained from three independent experiments. Error bars represent SD. For RNA blot analysis, RNA samples (20 μg) were denatured, fractionated on formaldehyde–agarose gels, transferred onto membranes and hybridized to the 32P-labeled cDNA inserts isolated from the corresponding plasmids. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel (bottom). Fig. 6 View largeDownload slide Effect of norflurazon on the accumulation of LLP-Rop1 and LLP-12-2 transcripts in developing anthers of L. longiflorum. Freshly cut lily plants with buds of 9.5 cm were dipped in an aqueous solution without or with the addition of 10 or 100 μM norflurazon and grown to reach anthesis (15.5-cm buds). Total RNA was isolated from anthers of various bud sizes. RNA levels of LLP-Rop1 and LLP-12-2 were determined by Q-PCR and RNA blot hybridization. For Q-PCR, the data were obtained from three independent experiments. Error bars represent SD. For RNA blot analysis, RNA samples (20 μg) were denatured, fractionated on formaldehyde–agarose gels, transferred onto membranes and hybridized to the 32P-labeled cDNA inserts isolated from the corresponding plasmids. LLA-23, a known dehydration- and ABA-induced gene (Huang et al. 2000) was used as a positive control. Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel (bottom). The ABA content in the developing pollen was further examined. As shown in Table 2, the ABA content detected in the pollen 1 d before anthesis (13.5- to 15.5-cm buds) was approximately 2-fold that of 9.5- to 10.5-cm pollen buds. The increase in ABA correlates with the desiccation that occurs in the maturing anther prior to anthesis. Table 2 ABA content in lily pollen at various maturation stages Bud size (cm)  ABA content (μg/g FW)  9.5–10.5  0.11 ± 0.02  13.5–15.5  0.19 ± 0.01  15.5–16.5  0.20 ± 0.01  Bud size (cm)  ABA content (μg/g FW)  9.5–10.5  0.11 ± 0.02  13.5–15.5  0.19 ± 0.01  15.5–16.5  0.20 ± 0.01  Data are mean ± SD of three individual experiments (n = 3 per experiment). View Large Overexpression of LLP-Rop1 in lily pollen tubes causes distinct tip growth phenotypes and subcellular localization To examine the function of LLP-Rop1, we tagged LLP-Rop1 with cyan fluorescent protein (CFP). CFP allowed both transformed pollen to be identified and the localization of CFP–LLP-Rop1 to be observed using confocal microscopy. LLP-Rop1 overexpression inhibited tube elongation and caused tube expansion and formation of a ballooned tip (Fig. 7B). As indicated in Table 3, LLP-Rop1 overexpression inhibited tube elongation by 60% and caused a >2-fold tube expansion when compared with overexpression of CFP only. While CFP–CA-llp-rop1, a constitutively active (CA) Q64L mutant of LLP-Rop1 caused enhanced inhibition of tube elongation and tip swelling, CFP–DN-llp-rop1, a dominantly negative (DN) D121A mutant of LLP-Rop1 exhibited reduced inhibition of pollen tube elongation (with a 37% reduction in length) but had little effect on pollen tube expansion (Table 3). Fig. 7 View largeDownload slide LLP-12-2 is a direct target of LLP-Rop1 GTPase. (A) Diagrammatic constructs of Zm13::CFP, Zm13::CFP–LLP-Rop1, Zm13::CFP–CA-llp-rop1, Zm13::CFP–DN-llp-rop1 and Zm13::YFP–LLP-12-2. Zm13, a pollen-specific strong promoter. (B) Subcellular localization of LLP-Rop1 and its mutants in pollen tips. Five rectangular areas starting from pollen tip to the center of the cytoplasm were randomly selected and each rectangular area was equally divided into three sections: outer, intermediate and inner. The intensity of fluorescence was measured by ImageJ software (http://rsbweb.nih.gov/ij/) and shown as artificial units. (C) FRET analysis of LLP-Rop1 interaction with LLP-12-2. Lily pollen tubes coexpressing CFP–LLP-Rop1, CFP–DN-llp-rop1 and YFP–LLP-12-2 were analyzed. A pseudocolor scale with the intensity of FRET signals and FRET efficiency (% of YFP acceptor emission) is displayed in the right panel (white, highest signal). All images were mid-plane confocal optimal sections. Bar = 20 μm. Fig. 7 View largeDownload slide LLP-12-2 is a direct target of LLP-Rop1 GTPase. (A) Diagrammatic constructs of Zm13::CFP, Zm13::CFP–LLP-Rop1, Zm13::CFP–CA-llp-rop1, Zm13::CFP–DN-llp-rop1 and Zm13::YFP–LLP-12-2. Zm13, a pollen-specific strong promoter. (B) Subcellular localization of LLP-Rop1 and its mutants in pollen tips. Five rectangular areas starting from pollen tip to the center of the cytoplasm were randomly selected and each rectangular area was equally divided into three sections: outer, intermediate and inner. The intensity of fluorescence was measured by ImageJ software (http://rsbweb.nih.gov/ij/) and shown as artificial units. (C) FRET analysis of LLP-Rop1 interaction with LLP-12-2. Lily pollen tubes coexpressing CFP–LLP-Rop1, CFP–DN-llp-rop1 and YFP–LLP-12-2 were analyzed. A pseudocolor scale with the intensity of FRET signals and FRET efficiency (% of YFP acceptor emission) is displayed in the right panel (white, highest signal). All images were mid-plane confocal optimal sections. Bar = 20 μm. Table 3 Phenotypic alteration of LLP-Rop1 and its mutants overexpressed in germinating pollen of lily plants   Length (μm)  Width (μm)  Width/length  CFP  300.07 ± 52.39  14.47 ± 0.31  0.05 ± 0.00  CFP–LLP-Rop1  119.47 ± 35.13  34.13 ± 7.23  0.29 ± 0.03  CFP–CA-llp-rop1  89.93 ± 25.60  35.40 ± 3.67  0.74 ± 0.49  CFP–DN-llp-rop1  188.27 ± 19.83  15.67 ± 1.86  0.08 ± 0.00    Length (μm)  Width (μm)  Width/length  CFP  300.07 ± 52.39  14.47 ± 0.31  0.05 ± 0.00  CFP–LLP-Rop1  119.47 ± 35.13  34.13 ± 7.23  0.29 ± 0.03  CFP–CA-llp-rop1  89.93 ± 25.60  35.40 ± 3.67  0.74 ± 0.49  CFP–DN-llp-rop1  188.27 ± 19.83  15.67 ± 1.86  0.08 ± 0.00  Pollen tube length and the tube diameter (width) at the most dilated region of the tube were measured 4 h after particle bombardment. Data are mean ± SD of three individual experiments (n = 25 per experiment). View Large To investigate the intracellular localization of LLP-Rop1, lily pollen tubes expressing CFP–LLP-Rop1 fusions were examined using confocal microscopy. As shown in Fig. 7B, CFP alone was distributed evenly in the pollen tube cytoplasm. CFP–LLP-Rop1, however, was localized to the cytoplasm having a greater intensity along the tube plasma membrane, a pattern correlated with the localization of LLP-12-2 (Hsu and Wang 2006). While the mutant CFP–CA-llp-rop1 was localized to the cytoplasm with enhanced intensity along the tube plasma membrane, CFP–DN-llp-rop1, in contrast, was primarily localized to the cytoplasm of pollen tubes similarly to CFP alone (Fig. 7B). The fluorescence of each CFP construct in the pollen was further quantified. The results revealed a significant distributive tendency of LLP-Rop1 along the tube plasma membrane. LLP-Rop1 interacts with LLP-12-2 protein during tube growth The LLP-12-2 protein was reported to be a RIC protein (Hsu and Wang 2006). RICs are proteins that share a CRIB motif, which is present in many Cdc/Rac effectors and responsible for their specific interaction with activated Cdc42/Rac (Wu et al. 2001). To examine whether LLP-12-2 is a target protein of LLP-Rop1, the coding region of LLP-12-2 was fused to that of yellow fluorescent protein (YFP) and the construct was driven by Zm13, a pollen-specific promoter (Fig. 7A). Pollen tube overexpressing LLP-12-2 (Hsu and Wang 2006) has a phenotype similar to that caused by LLP-Rop1 overexpression. Therefore, it is favorable to suggest that LLP-12-2, a lily RIC, may be the LLP-Rop1 target involved in the Rop signaling to control polarized tip growth in pollen tubes. If LLP-12-2 is the LLP-Rop1 target, it is expected to bind active LLP-Rop1 and to display LLP-Rop1 activation-dependent localization and function. In vivo LLP-Rop1 interaction with LLP-12-2 was tested using FRET analysis of lily pollen tubes coexpressing CFP–LLP-Rop1 and YFP–LLP-12-2 fusion genes. The FRET signal was obtained from YFP emission with excitation at 440 nm for CFP and corrected for non-FRET signals (see Materials and Methods for details). For comparison between treatments, FRET signals were normalized with the amount of acceptor and were presented as FRET efficiency (% of YFP emission resulting from 440-nm compared with 515-nm excitation). As shown in Fig. 7C, the YFP–LLP-12-2 signal was detected when tubes were excited with a laser that excites CFP but not YFP, indicating that FRET was occurring. No FRET signals were detected when CFP–LLP-Rop1 was replaced with CFP–DN-llp-rop1 (Fig. 7C). These results indicate that LLP-12-2 interacts with active LLP-Rop1 in vivo. Pollen exhibits no appreciable change in interaction between LLP-Rop1 and LLP-12-2 proteins upon dehydration To examine the change in interaction between LLP-Rop1 and LLP-12-2 proteins at the stage of desiccation, FRET analysis of lily pollen coexpressing CFP–LLP-Rop1 and YFP–LLP-12-2 fusion genes was used. Anthers of lily buds at anthesis were taken and dehydrated at various time intervals. The water content in the pollen dropped from 60% to <10% as pollen continued to air-dry for 48 h (Fig. 8A). FRET signals were normalized with the amount of acceptor and were presented as FRET efficiency. Using FRET analysis, the FRET efficiency of the outer section did not display an appreciable change in protein interaction between LLP-Rop1 and LLP-12-2 even in pollen air-dried for 48 h (Fig. 8B). This indicates that dehydration probably does not affect the interaction between LLP-Rop1 and LLP-12-2 proteins in the dried pollen. Fig. 8 View largeDownload slide Pollen exhibits no appreciable change in interaction between LLP-Rop1 and LLP-12-2 proteins upon dehydration. (A) Changes in percentage water content of pollen upon dehydration. (B) FRET analysis of LLP-Rop1 interaction with LLP-12-2 upon dehydration. Pollen taken from anthers of lily buds at anthesis was air-dried for various time intervals. FRET analysis of LLP-Rop1 interaction with LLP-12-2 was carried out in pollen tubes coexpressing CFP–LLP-Rop1 and YFP–LLP-12-2. A pseudocolor scale with the intensity of FRET signals and FRET efficiency (% of YFP acceptor emission) is displayed in the right panel (white, highest signal). All images were mid-plane confocal optimal sections. Bar = 20 μm. Five rectangular areas starting from pollen tip to the center of the cytoplasm were randomly selected, each of which was equally divided into three sections: outer, intermediate and inner. FRET efficiency was measured by ImageJ software (http://rsbweb.nih.gov/ij/) and shown as artificial units. Fig. 8 View largeDownload slide Pollen exhibits no appreciable change in interaction between LLP-Rop1 and LLP-12-2 proteins upon dehydration. (A) Changes in percentage water content of pollen upon dehydration. (B) FRET analysis of LLP-Rop1 interaction with LLP-12-2 upon dehydration. Pollen taken from anthers of lily buds at anthesis was air-dried for various time intervals. FRET analysis of LLP-Rop1 interaction with LLP-12-2 was carried out in pollen tubes coexpressing CFP–LLP-Rop1 and YFP–LLP-12-2. A pseudocolor scale with the intensity of FRET signals and FRET efficiency (% of YFP acceptor emission) is displayed in the right panel (white, highest signal). All images were mid-plane confocal optimal sections. Bar = 20 μm. Five rectangular areas starting from pollen tip to the center of the cytoplasm were randomly selected, each of which was equally divided into three sections: outer, intermediate and inner. FRET efficiency was measured by ImageJ software (http://rsbweb.nih.gov/ij/) and shown as artificial units. Discussion We report unique desiccation-associated ABA signaling through which the Rop gene is regulated in maturing and dried pollen. Using FRET analysis, we were able to determine that LLP-Rop1 indeed interacts with the LLP-12-2 protein in vivo. LLP-Rop1 and LLP-12-2, the target protein gene of LLP-Rop1, responded to desiccation via the ABA signaling transduction pathway. LLP-Rop1 and LLP-12-2 proteins each constitute a gene family, implying that various RIC proteins may coordinate to regulate distinct Rop-dependent pathways through their differential interactions with Rops during pollen maturation and tube growth (Wu et al. 2001, Christensen et al. 2003). The LLP-Rop1 and LLP-12-2 genes respond to both desiccation and other developmental cues during anther/pollen maturation Since both LLP-Rop1 and LLP-12-2 mRNAs accumulate to a maximum level as anthers grow to maturity, it is possible that the two genes might be associated with desiccation. Premature drying of young anthers confirms that transcripts of both genes are dehydration related. It should be stressed that the two genes respond to desiccation, a part of the developmental program that occurs naturally in the anther. Moreover, the two genes can also be induced by other developmental cues based on the observation that the ABA biosynthesis inhibitor, norflurazon, cannot completely diminish the accumulation of either LLP-12-2 or LLP-Rop1 transcript (Fig. 6). The retained transcripts of LLP-12-2 or LLP-Rop1 reflects that, in addition to desiccation, unknown factors (developmental cues) independent of those well characterized by ABA signaling are involved in gene regulation. Thus, a number of distinct pathways of signaling can be established based on their expression patterns at the phase of desiccation during anther/pollen maturation as previously described by Hsu et al. (2007). However, the fact that the LLP-Rop1 transcript eventually accumulates in abundance in the mature and dried pollen (Fig. 3B) suggests that the influence of developmental cues on expression of LLP-Rop1 gene is more prominent than the adverse effect caused by dehydration. The lesser effect caused by dehydration is also reflected by the fact that an increase in the level of LLP-Rop1 transcript is not discernible during development when norflurazon is applied (Fig. 6). While the LLP-Rop1 gene is upregulated by developmental cues, the same gene is downregulated by desiccation in the maturing pollen. This apparent contradiction strongly suggests the existence of a fine-tuning mechanism by which the LLP-Rop1 gene is regulated. This regulating system ensures that appropriate amounts of LLP-Rop1 gene product are obtained in the mature and dried pollen to follow germination and tube growth. The downregulation of the LLP-Rop1 gene by ABA may be an essential early signaling event to induce ABA response while ABA suppression of Rop gene transcription could be crucial for sustained ABA action. It correlates with the observation of an increase in ABA content in the maturing and dried pollen (Table 2). The regulatory mechanism correlates with the finding that Rop is a specific negative regulator of ABA signaling (Xin et al. 2005). Baxter-Burrell et al. (2002) have reported a different pattern of negative regulation of Rops where lack of oxygen is proposed to activate Rop signaling leading to the production of H2O2, which in turn increases the expression of alcohol dehydrogenase. At the same time, H2O2 induces the expression of the gene encoding RopGAP4, a member of the GTPase-activating protein (GAP) family that deactivates Rops by promoting hydrolysis of GTP. Thus, Rop activation of RopGAP4 expression functions as a negative feedback loop to terminate Rop signaling. At least four classes of Rho GTPase interactor are known to regulate Rho-family GTPases. Aside from two classes of activator, guanine nucleotide exchange factors (GEFs) and scaffolding proteins, there are two classes of molecule known to deactivate Rho-family GTPases: GAPs and guanine nucleotide dissociation inhibitors (GDIs) (Gu et al. 2004). GDIs downregulate Rho GTPases both by sequestering them in the cytosol and by suppressing their activation by GEFs (Fauré and Dagher 2001). It is worthy of mention that Arabidopsis possesses three RhoGDIs (Bischoff et al. 2000). In this report we observe unique desiccation-associated ABA signaling in which the LLP-Rop1 gene is not only negatively regulated by desiccation but also positively regulated by developmental cues independent of ABA during pollen maturation. Despite significant progress in the understanding of ABA biosynthesis (Xiong and Zhu 2003), the desiccation-associated ABA signaling pathway occurring in the anther has yet to be reported. The LLP-Rop1 and LLP-12-2 genes antagonistically respond to desiccation via the ABA signaling pathway It is surprising that premature drying analysis shows that the LLP-12-2 transcript increases while the LLP-Rop1 transcript decreases its level of accumulation (Fig. 4A). This relationship correlates with a similar antagonistic pattern of accumulation when exogenous ABA is applied either to young anthers (Fig. 4B) or to pollen during germination (Fig. 5). The ABA inhibition analysis suggests that the two genes are indeed responsive to endogenous ABA that presumably arose upon the occurrence of dehydration in the maturing pollen. The measurement of ABA content in the developing pollen confirms an increase in ABA immediately prior to anthesis (Table 2). Therefore, it is likely that desiccation occurs in the anther prior to anthesis inducing the production of ABA and subsequently altering gene expression. Our observation of decreased LLP-Rop1 mRNA level upon desiccation is consistent with an earlier report of AtROP10 transcription specifically downregulated by ABA (Zheng et al. 2002). Rop was newly reported as a master regulator for plant signaling (Gu et al. 2004, Berken 2006). It participates in concerted actions of many signaling pathways that influence growth and development, and the adaptation of plants to various environmental situations. Aside from being linked to various aspects of polar growth during tube elongation, Rops may regulate plant responses to microbes, secondary wall formation and may influence the fate of meristematic tissue (Trotochaud et al. 1999, Brembu et al. 2005, Szucs et al. 2006). Rop GTPases may be involved in salt tolerance and disease resistance in rice growth (Jung et al. 2006, Luo et al. 2006). Rop GTPases may also activate the levels of reactive oxygen species in the whole leaf and single cells of Arabidopsis (Park et al. 2004). In contrast to ABA signaling, Rops might work as positive regulators in auxin signaling pathways (Tao et al. 2005). Pollen is a unique organ in that it contains pre-synthesized mRNAs and proteins, which are the main products of those selectively expressed and enriched genes (Willing et al. 1988). The activity of LLP-Rop1 and LLP-12-2 is likely restricted in the dried pollen. Accordingly, it is reasonable to suggest that the subsequent interaction between LLP-Rop1 and LLP-12-2 would not occur at the dried stage of pollen maturation. Corroborant data were obtained using FRET analysis to examine the interaction of LLP-Rop1with LLP-12-2 in the pollen under treatments of various degrees of dehydration. No appreciable change in protein interaction between LLP-Rop1 and LLP-12-2 was observed when pollen was air-dried (Fig. 8). Thus, this indicates that dehydration probably does not affect the interaction between LLP-Rop1 and LLP-12-2 proteins in the dried pollen although dehydration affects the expression of the LLP-Rop1 and LLP-12-2 genes. It should be noted that pollen taken from anthers of lily buds prior to anthesis and subsequently subjected to particle bombardment gave poor germination and so pollen was tested from anthers of buds at anthesis. The abundance of pre-synthesized mRNAs of LLP-Rop1 and LLP-12-2 and their translated proteins is reserved for the subsequent use of pollen germination and tube growth. As pollen germinates, ABA content decreases its level in the growing tube and thus, the activity of Rop is less restricted than in the dried pollen. It correlates with the fact that Rop GTPase is a powerful regulator playing crucial roles during pollen tube growth. We report unique desiccation-associated ABA signaling transduction through which the LLP-Rop1 gene is regulated for likely sustained ABA action. The LLP-Rop1 and LLP-12-2 genes are expressed differently in response to both desiccation and other developmental cues in the developing pollen. Both genes are upregulated by other developmental cues but upon desiccation the LLP-Rop1 gene is downregulated while the LLP-12-2 gene is upregulated. Furthermore, using FRET analysis we demonstrate that the LLP-Rop1 protein interacts with LLP-12-2 in vivo. Materials and Methods Plant materials Lily (Lilium longiflorum Thunb. cv. Snow Queen) plants were grown in the field. Buds ranging from 1 to 17 cm were dissected to isolate anthers that varied in length from 0.7 to 2.3 cm. Meiosis occurred in the pollen mother cells at a bud size of around 2–2.5 cm, resulting in the formation of tetrads. Afterwards, microspore mitosis was complete at a bud length of around 6–7 cm and pollen subsequently entered the maturation phase of development. Dehydration occurs in anthers of 13.5- to 15.5-cm buds, prior to anthesis (15.5- to 16.5-cm buds). The anther was separated from the filament in buds longer than 2.5 cm; otherwise the two organs were combined. Pollen was manually collected 1 or 2 d after anthesis. The first three arrays of young leaves around buds, entire roots (approximately 8 cm from the apex) and stems were collected and frozen immediately in liquid nitrogen. Material was stored at −80°C until use. Premature drying, ABA and inhibitor and germination treatments For the analysis of premature drying, anthers of indicated flower bud sizes were taken and air-dried on the laboratory bench (relative humidity about 30%) for 30 h at room temperature. To investigate the influence of ABA and norflurazon (AccuStandard, Inc., New Haven, CT, USA), an ABA biosynthesis inhibitor, on gene expression during anther development, freshly cut lily plants with indicated buds sizes were dipped in aqueous solutions without (control) or with the addition of 10 or 100 μM ABA for 24 h. For the addition of norflurazon, freshly cut lily plants with buds of 9.5 cm were dipped in an aqueous solution not containing (control) or containing 10 or 100 μM norflurazon and grown to reach anthesis. Afterwards, anthers were collected from lily buds of the above treatments, immediately frozen in liquid nitrogen and stored at −80°C. For pollen germination, pollen (approximately 200 mg) was incubated with or without the application of 10 or 100 μM ABA in 10 ml of germination medium [0.29 M pentaerythritol, 300 μg ml−1 Ca(NO3)2·4H2O, 10 μg ml−1 H3BO3 and 100 μg ml−1 KNO3] (Dickinson 1978) at 30°C with shaking at the indicated time intervals. To avoid imbibitional damage, we routinely placed frozen or dried pollen in humid air for at least 1 h. RT–PCR A reaction mix (5 μl) consisting of 0.1 μg of total RNA and 0.5 μg of oligo(dT) (Promega, Madison, WI, USA) was denatured at 70°C for 2 min after which 1 μl of 10× first-stranded buffer [750 mM KCl, 30 mM MgCl2, 100 mM dithiothreitol, 500 mM Tris–HCl, pH 8.3], 1 μl of 10 mM dNTPs and 0.5 μl of 200 U μl−1 MMLV reverse transcriptase (New England BioLabs, Beverly, MA, USA) were consecutively added. The RT reaction mix was incubated at 42°C for 90 min, heated at 72°C for 2 min, diluted to give a final volume of 50 μl and kept at −20°C. RT–PCR analyses were performed using the gene-specific primer pairs as listed in Table 1. The PCR mix (50 μl) consisted of 1 μl of the RT reaction mix, 2.5 mM MgCl2, 0.2 mM dNTPs, 1 × PCR buffer (10 mM Tris–HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1 μM each of the primer pair and 2.5 U of Taq DNA polymerase (MD Bio. Inc., Taipei, Taiwan). The reaction was conducted under the following protocol: 94°C for 10 min; 25 cycles at 94°C for 1 min, annealing at 56°C for 1 min, 72°C for 1 min; final elongation at 7°C for 10 min in the PTC-200 Peltier thermal cycler (MJ Research, Watertown, MA, USA). The resulting PCR products were run on a 1.5% agarose gel containing 0.01% ethidium bromide. The housekeeping 18S ribosomal gene with a fragment of 452 bp was used as a positive control for PCR amplification. RNA blot analysis Total RNA was extracted from floral and vegetative organs, developing anthers with various treatments, and germinating and ungerminating pollen according to the method of Verwoerd et al. (1989). For RNA blot analysis, RNA samples were electrophoresed in 1.0% formaldehyde/3-[N-morpholino]propanesulfonic acid agarose gels and transferred onto nylon membranes (Immobilon-Ny+ membrane; Millipore, Bedford, MA, USA). The membranes with immobilized RNA were prehybridized for 4 h at 42°C in medium containing 5× SSC (0.75 M NaCl and 75 mM sodium citrate, pH 7.0), 0.1% polyvinylpyrrolidone, 0.1% Ficoll, 20 mM sodium phosphate, pH 6.5, 0.1% (w/v) SDS, 1% glycine, 50% formamide and 150 μg ml−1 of denatured salmon sperm DNA using standard procedures (Sambrook et al. 1989). For hybridization, the prehybridization solution was removed and replaced with hybridization buffer that contained the same components as the prehybridization buffer but lacked 1% glycine. Denatured salmon sperm DNA (100 μg ml−1) and random-primed 32P-labeled probe (1.0 × 109 cpm μg−1) were added to the hybridization buffer. Hybridization was carried out at 42°C overnight with constant agitation. The membranes were washed twice at 50°C in 2× SSC, 0.1% (w/v) SDS for 20 min, followed by twice at 60°C in 0.1× SSC, 0.1% (w/v) SDS for 20 min. The membrane was visualized using phosphoimager plates using either one or two intensifying screens (DuPont) for 3 d or less. Quantitative RT–PCR For real-time Q-PCR, the cDNA was amplified in the presence of SYBR Green I nucleic acid gel stain (Lonza) 10 000× dilution from stock using a Rotor-Gene 3000 (Corbett). Amplification of rRNA under identical conditions was used as an internal control to normalize the level of cDNA. The data obtained were analyzed with Rotor-Gene 6 software (Corbett). Since SYBR Green I dye binds to the minor groove of any double-stranded DNA, including specific products, non-specific products and primer dimers, it is necessary to perform a melting curve analysis at the end of each Q-PCR experiment. Non-specific products or primer dimers can be identified as they melt at a lower temperature compared with the specific amplicon. Specific temperatures obtained for rRNA (64°C), LLP-Rop1 (53°C) and LLP-12-2 (64°C) validated the specific product formation. Q-PCR experiments were repeated three times independently and samples in each experiment were made in duplicate. Measurement of ABA content ABA measurement is according to the method of Xiong et al. (2001). Briefly, 0.2 g of the frozen pollen was suspended in 3 ml of extraction solution containing 80% methanol, 100 mg l−1 butylated hydroxytoluene and 0.5 g l−1 citric acid monohydrate. The suspension was stirred overnight at 4°C and centrifuged at 1000 × g for 20 min. The supernatant was transferred to a new tube, dried under vacuum and dissolved with 20 ml of methanol and 180 ml of Tris-buffered saline (50 mM Tris, 0.1 mM MgCl2·6H2O and 0.15 M NaCl, pH 7.8). ABA concentration in the solution was determined using the Phytodetek ABA Test Kit (Agdia, Elkhart, IN, USA). Site-directed mutagenesis in vitro PCR-based site-directed mutagenesis was used to create point mutation of LLP-Rop1. PCR was used to amplify the constitutively active (CA) mutant, CA-llp-rop1 (Q64L) with a pair of mutagenesis primers: CA-llp-rop1-f and CA-llp-rop1-r (Table 1) and the pGEM-T Easy Vector containing LLP-Rop1 as a template. The reaction mixture (50 μl) is composed of 1 × Pfu buffer [20 mM Tris–HCl, pH 8.8, 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100], 2.5 mM MgCl2, 0.2 mM dNTPs and 5 U of Pfu polymerase (Stratagene, La Jolla, CA, USA), 1 mM each of the mutagenesis primers and 1 ng of recombinant pGEM-T Easy Vector. The reaction was conducted under the following protocol: 94°C for 10 min; 32 cycles at 94°C for 1 min, annealing at 65°C for 1 min, elongation at 72°C for 1 min; final elongation at 72°C for 10 min. The 4.0-kb PCR product was purified from 1.0% agarose gel and treated with DpnI at 37°C for 1 h. The DpnI-treated DNA product was fractionated, eluted and self-ligated at 4°C overnight, in 20 μl of solution containing 1× rapid ligation buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP) and 3 U of T4 DNA ligase (Promega). The CA-llp-rop1 mutant was selected and confirmed by DNA sequencing. The dominantly negative (DN) mutant, DN-llp-rop1 (D121A) was obtained in a fashion similar to CA-llp-rop1 except that the mutagenesis primer pair DN-llp-rop1-f and DN-llp-rop1-r was used (Table 1) instead of CA-llp-rop1-f and CA-llp-rop1-r. Particle bombardment-mediated transient expression in pollen PCR was first used to amplify LLP-Rop1 with a pair of primers: EcoRI–LLP-Rop1-f and LLP-Rop1–SacI-r (Table 1) and LLP-Rop1 as a template. The primers were designated as such because the EcoRI–LLP-Rop1-f primer contains an EcoRI site followed by the start codon of LLP-Rop1 and the LLP-Rop1–SacI-r primer contains the stop codon and SacI. Next, the resulting PCR product digested with EcoRI and SacI was ligated with the CFP gene digested with BamHI and EcoRI in order to generate a CFP–LLP-Rop1 fusion. The constructs CFP–CA-llp-rop1 (Q64L) and CFP–DN-llp-rop1 (D121A) were made according to the same procedure except that CA-llp-rop1 cDNA or DN-llp-rop1 was used as a template instead of LLP-Rop1 when PCR amplification was performed. The fusion with BamHI and SacI at either end was then cloned into the corresponding sites of pZm13 vector, which includes the Zm13 promoter (a gift from Dr Tanaka I, Yokohama City University, Yokohama City, Japan). The CFP alone was also amplified by PCR using a pair of primers: BamHI–CFP-f and CFP–SacI-r (Table 1) and pECFP as a template. The 5′-primer contains the start codon and BamHI and the 3′-primer contains a termination codon and SacI. The nucleotide sequence of each fusion construct was verified by DNA sequencing, A Zm13::YFP–LLP-12-2 fusion was also generated. The YFP gene with a BamHI site at the 5′ and an EcoRI site at the 3′ end was amplified by PCR using a pair of primers: BamHI–CFP-f and CFP–EcoRI-r and pEYFP vector as a template. A parallel construct of LLP-12-2 fragment with an EcoRI site at its 5′ end and a SacI site at its 3′ end was obtained by PCR with a pair of primers: EcoRI–LLP-12-2-f and LLP-12-2–SacI-r and pM12N containing LLP-12-2 cDNA as a template (Hsu and Wang 2006). The EcoRI–LLP-12-2-f primer contains an EcoRI site followed by the start codon of LLP-12-2 while the LLP-12-2–SacI-r primer contains the stop codon of LLP-12-2 followed by a SacI site. The purified LLP-12-2 digested with EcoRI was ligated with EcoRI-digested YFP. The resulting YFP–LLP-12-2 fusion was then digested with BamHI and SacI and inserted into pZm13 vector to generate the Zm13::YFP–LLP-12-2 construct. The construction of Zm13::YFP was the same as that of Zm13::CFP except that pEYFP vector was used as a template when PCR amplification was conducted. Particle bombardment-mediated transient expression in lily pollen was modified using PDS-1000/He Biolistic Particle Delivery System (Bio-Rad, Hercules, CA) (Chen et al. 2002). Pollen grains (8 mg) were rehydrated for 1 h and sprayed onto germination medium-immersed filter paper in a 9-cm Petri dish. Routinely, 0.77 mg of 1.0 Micron Gold (Bio-Rad) was coated with 3.6 μg of DNA of each corresponding plasmid or, for FRET analysis, 1.8 μg of Zm13::CFP–LLP-Rop1 or Zm13::CFP–DN-llp-rop1 was mixed with an equal amount of Zm13::YFP–LLP-12-2. The gold-coated plasmids were split into three equal parts for bombardment. The parameters of bombardment were as follows: 1100-p.s.i. rupture disc, 29-mm Hg vacuum, 1-cm gap distance and 9-cm particle flight distance. Bombarded pollen grains were washed into a 3.5-cm Petri dish with 2 ml of germination medium and incubated with gentle shaking at 30°C for 6 h. FRET analysis For FRET analysis, pZm13::CFP–LLP-Rop1 was transiently co-overexpressed with pZm13::YFP–LLP-12-2 in lily pollen. FRET analysis was performed 6 h after particle bombardment using a confocal microscope (Olympus, FV1000) equipped with a He–Cd laser (used for CFP excitation at 440 nm) and an argon laser (which provides excitation at 515 nm for YFP). The spectral photometric detection system of the FV1000 confocal device allows freely adjusting collection bandwidth for emission signal to minimize bleedthrough signals. Settings for these experiments were as follows: (a) CFP channel: used CFP excitation at 440 nm, emission from 470 to 500 nm detected CFP–LLP-Rop1; (b) YFP channel: used YFP excitation at 515 nm, emission from 535 to 600 nm for the detection of YFP–LLP-12-2; and (c) FRET channel: used CFP excitation at 440 nm, emission from 535 to 600 nm for the detection of YFP, equivalent to FRET signal. CFP and YFP images were simultaneously obtained to show localization of LLP-Rop1 and LLP-12-2 prior to the acquisition of FRET images at the same confocal plane. The FRET signal was adjusted by subtraction of YFP emission due to non-FRET that includes CFP bleedthrough to the FRET channel and YFP acceptor excitation by 440-nm laser. Correction factors for non-FRET signals were calculated from CFP-donor-alone and YFP-acceptor-alone controls. Corrected FRET signal was normalized with the acceptor amount and shown as FRET efficiency (i.e. % of YFP emission resulting from 440-nm against 515-nm excitation). The intensity of FRET signals and FRET efficiency were calculated and shown as a pseudocolor scale. Determination of water content in pollen grains Pollen removed from anthers of lily buds at anthesis was air-dried on the laboratory bench for various lengths of time at 25°C. The fresh and air-dried pollen were then placed in a forced air oven at 60°C for 2 d. Data were obtained from three independent experiments. Water content was determined by measuring the difference before and after oven treatment and expressed as the percentage of the fresh weight. Funding This work was supported by the National Science Council, Taiwan (grant number NSC 98-2321-B005-003-MY3 to C.-S.). Abbreviations Abbreviations ABA abscisic acid CA constitutively active CFP cyan fluorescent protein CRIB motif Cdc42/Rac-interactive binding motif DN dominantly negative FRET fluorescence resonance energy transfer PCR polymerase chain reaction PM plasma membrane RIC Rop-interactive CRIB motif-containing protein Rop Rho GTPase of plants RT reverse transcriptase YFP yellow fluorescent protein. The reported nucleotide sequence of LLP-Rop1 cDNA appears in the GenBank Databases under the accession number EF140700. 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TI - Rop GTPase and Its Target Cdc42/Rac-Interactive-Binding Motif-Containing Protein Genes Respond to Desiccation during Pollen Maturation
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcq076
DA - 2010-05-20
UR - https://www.deepdyve.com/lp/oxford-university-press/rop-gtpase-and-its-target-cdc42-rac-interactive-binding-motif-74TFX8oCfm
SP - 1197
EP - 1209
VL - 51
IS - 7
DP - DeepDyve
ER -