AtRabD2b and AtRabD2c have overlapping functions in pollen development and pollen tube growth
© Peng et al; licensee BioMed Central Ltd. 2011
Received: 3 September 2010
Accepted: 26 January 2011
Published: 26 January 2011
Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling. Many Rab-like proteins exist in plants, but only a subset have been functionally characterized.
Here we report that AtRabD2b and AtRabD2c play important roles in pollen development, germination and tube elongation. AtrabD2b and AtrabD2c single mutants have no obvious morphological changes compared with wild-type plants across a variety of growth conditions. An AtrabD2b/2c double mutant is also indistinguishable from wild-type plants during vegetative growth; however its siliques are shorter than those in wild-type plants. Compared with wild-type plants, AtrabD2b/2c mutants produce deformed pollen with swollen and branched pollen tube tips. The shorter siliques in the AtrabD2b/2c double mutant were found to be primarily due to the pollen defects. AtRabD2b and AtRabD2c have different but overlapping expression patterns, and they are both highly expressed in pollen. Both AtRabD2b and AtRabD2c protein localize to Golgi bodies.
These findings support a partially redundant role for AtRabD2b and AtRabD2c in vesicle trafficking during pollen tube growth that cannot be fulfilled by the remaining AtRabD family members.
Ras-like small GTP-binding proteins (GTPases) regulate diverse processes in eukaryotic cells including signal transduction, cell proliferation, cytoskeletal organization and intracellular membrane trafficking. GTPases are activated by GTP binding and inactivated by subsequent hydrolysis of bound GTP to GDP, thus acting as molecular switches in these processes [1, 2]. The Rab GTPase family is the largest and most complex within the Ras protein superfamily. Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling processes in eukaryotic cells [3–6]. Rab GTPase functions have been extensively studied in yeast and mammalian systems. Both in vivo and in vitro experiments have demonstrated that different Rab proteins function in distinct intracellular membrane trafficking steps and they are hypothesized to work together with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to promote specificity of vesicle transport to target compartments and facilitate vesicle and target membrane fusion [7–13]. They are therefore essential for the transport of proteins and membrane through the endomembrane system to their destination.
The Arabidopsis thaliana genome encodes 93 putative Ras superfamily proteins. Fifty-seven of these are Rab GTPases, more than in yeast but similar to the number in humans [13, 14]. According to their sequence similarity and phylogenetic clustering with yeast and mammalian orthologs, these Rab proteins were assigned to eight subfamilies, AtRabA to AtRabH, which can be further divided into 18 subclasses . Relatively few of the plant Rab orthologs have been investigated functionally. Most of these studies have used constitutively active (CA) and/or dominant negative (DN) mutations, generated by direct mutation of the conserved domain to restrict mutant GTPase proteins to the active GTP-bound form (constitutively active) or inactive GDP-bound form (dominant negative). Expression of CA or DN Rab GTPases can perturb the activity of the endogenous Rab, revealing their functional significance. For a number of plant Rab GTPases, expression of their CA and DN mutants in transformed plants, together with protein localization information, has shown that these Rabs perform functions similar to those of their yeast and mammalian orthologs [15–19].
Several reports indicate that Rab proteins are important for elongation of tip-growing cells in plants. For example, AtRabA4b is reported to localize to the tips of root hair cells and was proposed to regulate membrane trafficking through a compartment involved in the polarized secretion of cell wall components . NtRab2 GTPase is important for trafficking between the endoplasmic reticulum and Golgi bodies in tobacco pollen tubes and may be specialized to optimally support the high secretory demands in these tip growing cells . NtRabA (Rab11) in tobacco is predominantly localized to an inverted cone-shaped region at the pollen tube tip, and both constitutively active and dominant negative mutants resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility .
There are four genes in the Arabidopsis RabD subfamily, AtRabD1 (At3g11730), AtRabD2a (At1g02130, AtRab1b), AtRabD2b (At5g47200, AtRab1a) and AtRabD2c (At4g17530, AtRab1c) . In mammals, the orthologs of AtRabD, Rab1 isoforms, physically associate with the ER, ER-Golgi intermediate compartment and Golgi and regulate membrane trafficking between the ER and Golgi complex . Fluorescent protein fusions with AtRabD1, AtRabD2a and AtRabD2b localize to the Golgi and trans-Golgi network [22, 23], and transient expression in plant cells of dominant negative mutants of rabD2a or rabD1 resulted in the inhibition of ER-to-Golgi trafficking [15, 22, 24], suggesting a related function for the plant Rab1 homologs. Pinheiro et al.  isolated T-DNA insertion mutants in each of the AtRabD family genes and reported that each of the single and double mutants lacked a detectable phenotype. By contrast, a rabD2a rabD2b rabD2c triple mutant was lethal and a rabD1 rabD2b rabD2c triple mutant had stunted growth and low fertility, indicating that these gene family members perform important and overlapping functions.
We previously hypothesized that closely related genes with a high Pearson correlation in their RNA accumulation level are functionally redundant, and showed that expression patterns of both the AtRabD2b and AtRabD2c genes are negatively correlated with the process of starch synthesis , whereas the expression patterns of the remaining RabD genes are not. We therefore predicted that these two Rab proteins may have redundant functions that are not shared by the other two AtRabD family members. Here we show that AtRabD2b and AtRabD2c are highly correlated in their RNA accumulation level across a variety of experimental conditions. Phenotypic analysis of knockout mutants indicates that they are at least partially functionally redundant, and are important in pollen development and pollen tube growth. The proteins both localize to the trans-Golgi, consistent with their proposed role in trafficking from the ER to the Golgi apparatus.
The expression patterns of AtRabD2b and AtRabD2care closely correlated
Pearson correlation between expression patterns of AtRabD family members.
Identification of Null Mutations in the Genes AtRabD2b and AtRabD2c
Siliques Are Shorter in the AtrabD2b/2cDouble Mutant than in Either Single Mutant or in Wild-Type Lines
To evaluate phenotypes associated with the AtrabD2b and AtrabD2c mutants, homozygous AtrabD2b (three alleles, AtrabD2b-1, AtrabD2b-2 and AtrabD2b-3), AtrabD2c and AtrabD2b/2c mutants, along with wild-type siblings, were grown on agar plates with or without various hormone, nutrient and light treatments. We tested over 50 of the conditions described in the Gantlet website (http://www.gantlet.org); however, no significant phenotypic differences were observed in the seedlings for any of the mutant alleles (data not shown). In addition, we tested the seedling phenotype on media with or without sucrose or vitamin B5 and, consistent with previous reports , no obvious phenotypes were observed.
Complementation of AtrabD2b/2cMutant Phenotype
AtrabD2b/2c, AtrabD2b and AtrabD2cPollen Have Defects in Morphology and Pollen Tube Elongation
We originally identified AtRabD2b and AtRabD2c because the transcript accumulation patterns of these two genes correlate with those of many genes associated with starch metabolism. Indeed, the AtrabD2b/2c double mutant pollen stained less intensely with IKI than wild-type pollen (Figure 5C), suggesting a decreased starch content in the AtrabD2b/2c mutant pollen. This is consistent with the expression correlation, although the reason for this phenotype is unclear.
Pollen and Pollen Tube Defects Cause the Shorter Siliques in the AtrabD2b/2cMutant
In silico and GUS Analysis of AtRabD2b and AtRabD2cExpression
Subcellular localization of AtRabD2b and AtRabD2c
Rab GTPases are critical players in the transport of materials through the endomembrane system, controlling exocytosis of proteins and cell wall materials, endocytosis of receptors and transporters, and membrane recycling processes. Together with SNARE proteins, they promote specificity of vesicle transport to target compartments, ensuring that vesicles fuse only with their appropriate target and thus maintaining the distinct identity of individual organelles. Two Rab subfamilies, Rab1 (AtRabD orthologs) and Rab2 (AtRabB orthologs), have been reported to function in membrane trafficking between the ER and Golgi in mammalian cells [21, 28–34]. The plant Rab1 and Rab2 homologs AtRabD1, AtRabD2a and NtRab2 have also been reported to function in ER to Golgi vesicle transport [15, 16]. Here, we demonstrate a distinct physiological role for the Rab1 homologs AtRabD2b and AtRabD2c in pollen development and pollen tube growth.
Using the bioinformatics tool MetaOmGraph (http://www.metnetdb.org) [25, 26] to determine the pairwise Pearsons correlation value between the expression patterns of all of the 57 AtRab genes (Additional file 1, Table S1), we found that among the four AtRabDs, only the expression of AtRabD2b and AtRabD2c are highly correlated. From this data, we hypothesized that AtRabD2b and AtRabD2c have partially redundant functions that are not shared by the remaining AtRabD family members. To test our hypothesis, we used T-DNA insertion single and double mutants to confirm that AtRabD2b and AtRabD2c have functional overlap and show that they are both required for normal pollen development and tip growth of pollen tubes. We also showed that they both co-localize with the trans-Golgi marker ST-YFP, consistent with their proposed role in Golgi trafficking.
The conclusion that AtRabD2b and AtRabD2c are partially functionally redundant is based on several lines of evidence. First, although single mutant plants containing AtrabD2b or AtrabD2c mutant alleles are indistinguishable morphologically from their wild-type counterparts, even when grown under a variety of growth conditions, the AtrabD2b/2c double mutant has a short-silique phenotype. Second, both AtrabD2b and AtrabD2c mutant plants produce a small percentage of deformed and collapsed pollen grains, while AtrabD2b/2c lines produce a higher percentage of deformed pollen grains, many of which are severely deformed, some lacking nuclei. It is probable that such aberrant pollen would give rise to defects in pollen germination, and indeed, though the germination rate is similar between AtrabD2b or AtrabD2c single mutants and wild-type plants, about 10% of the pollen grains from AtrabD2b/2c double mutant plants are unable to geminate. Furthermore, AtrabD2b and AtrabD2c mutant pollen tubes do not grow apically as well as do wild-type pollen tubes and tend to have swollen tips and a shorter length; this phenotype is substantially more severe in AtrabD2b/2c double mutants. In addition, some pollen tubes from AtrabD2b/2c double mutants branch or burst, which is not seen in pollen tubes of wild-type plants or either single mutant. These data also indicate that the loss of function of the AtRabD2b/2c genes cannot be compensated for by the AtRabD1 or AtRabD2a genes, suggesting that either some function(s) of the AtRabD2b and AtRabD2c proteins are distinct from those of AtRabD1 or AtRabD2a, or that they are not expressed in the same cell types.
Both AtRabD2b and AtRabD2c co-localize with the Golgi marker ST-YFP upon transient expression in Arabidopsis leaf protoplasts, as was reported also for AtRabD2a (formerly called AtRab1b) . It is therefore possible that AtRabD2b and AtRabD2c function in vesicle trafficking between the ER and Golgi apparatus, as does AtRabD2a [15, 22]. Complete disruption of Rab function in ER-to-Golgi trafficking is expected to be lethal, due to loss of plasma membrane, vacuole and cell wall assembly and integrity. However, the AtrabD2b/2c double mutant is indistinguishable from wild-type plants, except for shorter siliques due to the pollen and pollen tube defects. There are several possible explanations for this. First, other AtRabs must perform the same function in vegetative tissue. The most likely candidates are AtRabD2a and AtRabD1, which could compensate for the loss of function in the AtrabD2b/2c mutant in most cell types . Moreover, other Rab families, such as tobacco RabBs (NtRab2s) have also been shown to be regulators of membrane trafficking between the ER and Golgi apparatus . AtRabBs (AtRab2s) may have the same function, such that they also participate in ER to Golgi vesicle trafficking. The pattern of AtRabB1b RNA accumulation is most highly correlated with that of AtRabD2b (67%) and AtRabD2c (68%) (Additional file 1, Table S1). These genes might compensate in part for the loss of function of AtrabD2b/2c.
Second, pollen tubes grow very rapidly compared with many other cell types. Pollen tubes elongate by tip growth, whereby the pollen cytoplasm is confined to the most proximal region of the tube, and growth is restricted to the tube apex . In vitro, lily pollen tubes grow at about 150 nm/sec  and Arabidopsis pollen tubes at 37 nm/sec ; in vivo, tobacco pollen can grow at 42 nm/sec . This fast growth is contingent on rapid vesicle trafficking to deliver large amounts of membrane and cell wall components to the apical region of the tubes. This extensive trafficking requirement may preclude the remaining Rabs from completely compensating for loss of AtRabD2b and AtRabD2c.
Third, computational analysis of public microarray data, together with studies of the expression pattern directed by the AtRabD2b and AtRabD2c promoters, indicated that both are widely expressed in most organs and several cell types, with high expression in pollen. Root hairs also showed expression of AtRabD2b, and, like pollen tubes, root hairs elongate by tip growth. However, root hair growth in the AtrabD2b/2c double mutant is indistinguishable from that of wild-type plants. This is consistent with the idea that AtRabD2b and AtRabD2c are required for vesicle trafficking in multiple cell types, and that the highest demand for this process may be in pollen and pollen tubes, in order to optimally support the large secretory requirement of these very rapidly elongating cells. In combination, these data indicate that the high expression of AtRabD2b and AtRabD2c in pollen may be important to facilitate membrane trafficking needed for pollen tube growth.
In summary, we used a T-DNA insertion mutant approach to demonstrate the function of AtRabD2b and AtRabD2c. Our data indicated that both are Golgi residents; they have similar but not identical expression patterns, but are both highly expressed in pollen; they are both involved in tip growth of pollen tubes; and they are at least partially functionally redundant. Future work will focus on elucidating the molecular basis for the pollen phenotype in the AtrabD2b/2c double mutant.
Plant Materials and Growth Conditions
Wild-type Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0), AtrabD2c-1, AtrabD2b-1 and AtrabD2b/2c (crosses of AtrabD2b-1 and AtrabD2c-1) mutants in the same genetic background were used. Seeds were sown in Sunshine Soil mix, incubated at 4°C for 2 to 3 days, then grown at 22°C, 70% relative humidity, in a 16-h light/8-h dark photoperiod .
Screening for T-DNA insertion mutants
Primers used in this study
Crossing and screening for double mutant
Single mutant alleles (AtrabD2b-1 and AtrabD2c-1; Figure 1A) were crossed, the F1 generation of these crosses was allowed to self fertilization and the AtrabD2b/2c double mutant was identified from the F2 generation by PCR genotyping.
Semi-quantitative reverse transcription PCR
Total RNA was extracted from leaves of 20 DAI (days after imbibition) plants using the TRIZOL reagent (Invitrogen). RT-PCR was performed using SuperScript™ III One-Step RT-PCR System (Invitrogen,) as per the manufacturer's manual. The β-tubulin gene, which is highly conserved and constitutively expressed in all eukaryotes, was used as a standard. The primers used are listed in Table 2. The RT-PCR products were sequenced to confirm the correct amplification product.
In vitropollen germination and growth measurement
Pollen was obtained from flowers collected from Arabidopsis plants (ten plant lines per genotype) 1 to 2 weeks after bolting. Pollen from AtrabD2b/2c, AtrabD2b and AtrabD2c mutants, along with pollen from wild-type plants, was germinated on agar medium containing 18% (w/v) sucrose, 0.01% (w/v) boric acid, 1mM MgSO4, 1mM CaCl2, 1mM Ca(NO3)2, and 0.5% (w/v) agar, pH 7.0  overnight at room temperature and examined and photographed under a Zeiss Axioplan II compound microscope equipped with an AxioCam color digital camera. Measurements were performed using SIS Pro software (OSIS, Lakewood, CO) using the bars in the original image. For pollen tube length measurements, 200 pollen tubes were chosen randomly for each genotype, and significance was assessed using Student's t-test.
For fluorescence microscopy, the germinated pollen was transferred onto a slide and two drops of aniline blue solution (0.005% aniline blue solution in 0.1 M sodium phosphate, pH 7.0) were added for ten minutes.
To confirm the pollen tube growth defects, 20 open flowers per genotype were cut below the pistil and inserted vertically into germination medium in a 9-cm Petri dish. Plates were sealed and incubated overnight at 22°C at 100% humidity under continuous illumination. The paths of pollen tubes inside the pistils were visualized by fixing whole pistils in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, under low vacuum (18 psi Hg) for 2 h at room temperature. Samples were washed three times in the same buffer and stained with Aniline Blue and DAPI. The tissue was then cleared for 24 hours at room temperature with a drop of clearing solution (240 g of chloral hydrate and 30 g of glycerol in 90 ml water). Pollen was examined with a Zeiss Axioplan 2 light microscope (LM) and images were captured with a Zeiss AxioCamHRc digital camera (Carl Zeiss,Inc., Thornwood, NY) using AxioVision 4.3 software. The microscope was equipped with a DAPI filter set comprising an excitation filter (BP 365/12 nm), a beam splitter (395 nm), and an emission filter (LP 397 nm). The objectives used for imaging were a Neofluar 40× oil, an Apochromat 63× oil, and a Neofluar 100× oil.
Promoter::GFP/GUS fusion constructs were made for each gene by cloning the amplified promoter region (intergenic region; 964 bp for AtRabD2b and 558 bp for AtRabD2c) into the binary vector pBGWFS7 (GATEWAY; Invitrogen).
The genomic fragments containing AtRabD2b or AtRabD2c with their respective promoters for complementation of the mutant phenotype were amplified using AtRabD2b-g-F and R or AtRabD2c-g-F and R primers (Table 2). Products were cloned into the pENTR/D vector (Invitrogen), and then were transferred into the pMDC123 binary vector for plant transformation.
Plant transformation and selection
Arabidopsis plants were transformed using Agrobacterium tumefaciens by the floral dip method  and selected for Basta resistance conferred by the T-DNA.
Transgenic T2 seedlings were germinated in soil and harvested at various stages of development. Plants or organs were stained at room temperature overnight as described , then destained in 70% (v/v) ethanol. For each construct, at least 7 independently transformed lines, 7 plants for each stage, were harvested for GUS screening.
Transient expression in protoplasts
Transient gene expression in Arabidopsis mesophyll protoplasts was carried out as described previously . In brief, Arabidopsis protoplasts were isolated from the leaves of 3-4 week old plants. Leaf strips were digested in a buffer containing cellulose R-10 and macerozyme R-10. After adding 30 μg of plasmid DNA, an equal volume of protoplasts was mixed with PEG buffer (40% (w/v) PEG4000, 25% (v/v) 0.8M mannitol, 10% 1M CaCl2) then incubated at room temperature for 25 min. After gentle washing, the protoplasts were kept in the dark at room temperature overnight and then viewed by confocal laser scanning microscopy as described below.
Confocal laser scanning microscopy
Colocalization of GFP-RabD2b and GFP-RabD2c with ST-YFP was performed using a Leica TCS SP10 confocal microscope, which allows flexible selection of emission bandwidths to minimize bleed-through. Transformed cells were excited with a 488 nm laser (power 20%) and 514 nm laser (50% power), and GFP and YFP signals were collected using 495-510 nm and 560-640 nm bandwidths, respectively. Non-transformed cells and cells expressing a single GFP or YFP fusion were used as controls to confirm the absence of cross talk between GFP, YFP and autofluorescence signals.
Scanning electron microscopy
Pollen that had been germinated in vitro was placed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, under low vacuum (18 psi Hg) for 5 h at room temperature. Samples were washed three times in the same buffer, postfixed in 1% osmium tetroxide in the same buffer for 2 h and washed two times in the same buffer, followed by deionized water. Samples were dehydrated through a graded ethanol series (50, 70, 85, 95, and 100%; 30 min per step), followed by two changes of ultrapure 100% ethanol, all 30 min per step. Fresh pollen was also examined without fixing. Fixed samples were critical point-dried in a DCP-1 Denton critical-point-drying apparatus (http://www.dentonvacuum.com) using liquid carbon dioxide, and mounted on aluminum stubs with double-sided sticky pads and silver cement.
Samples were then sputter-coated with 15 nm gold (20%) and palladium (80%) in a Denton Vacuum LLC Desk II Cold Sputter Unit (http://www.dentonvacuum.com), and viewed with a JEOL 5800LV SEM (http://www.jeol.com) at 10 kV. Alternatively, released fresh pollen grains were directly mounted on stubs and sputter-coated with gold particles before SEM analysis. All digitally collected images including the LM and SEM images were processed in Adobe PhotoShop 7.0 and made into plates using Adobe Illustrator 10. Over 20 samples from each plant line were used for SEM or LM analysis.
We are grateful to Ian Moore, University of Oxford, United Kingdom for kindly providing the N-ST-YFP construct and for helpful suggestions about the Rab genes. We also thank the Arabidopsis Biological Resource Center and the Salk Institute Genomic Analysis Laboratory for providing T-DNA insertion mutants. This research was supported in part by grant MCB-0951170 from the National Science Foundation to ESW and grant no. NNX09AK78G from the National Aeronautics and Space Administration to DCB.
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