These authors contributed equally to this work.
Transgenic expression of a putative calcium transporter affects the time of Arabidopsis flowering
Article first published online: 16 JAN 2003
The Plant Journal
Volume 33, Issue 2, pages 285–292, January 2003
How to Cite
Wang, D., Xu, Y., Li, Q., Hao, X., Cui, K., Sun, F. and Zhu, Y. (2003), Transgenic expression of a putative calcium transporter affects the time of Arabidopsis flowering. The Plant Journal, 33: 285–292. doi: 10.1046/j.1365-313X.2003.01627.x
- Issue published online: 16 JAN 2003
- Article first published online: 16 JAN 2003
- Received 12 August 2002; revised 7 October 2002; accepted 11 October 2002.
- calcium fluorescence;
- gene expression;
- Arabidopsis transformation;
- Pisum G2
PPF1 is a gibberellin-induced, vegetative growth-specific gene, first isolated from short-day (SD)-grown G2 pea plants. In the current work, we found that transgenic Arabidopsis plants overexpressing the PPF1 gene (PPF1 (+)) flowered much later and had a significantly longer lifespan compared to control plants, whereas suppression of this gene (PPF1 (–)) resulted in a very rapid reproductive cycle. Western blotting analyses of PPF1 (+) and (–) plant lines revealed a positive correlation between the amount of antibody-reactive protein and the time of flowering. Green flourescent protein (GFP) co-expression assays showed that the PPF1 protein is likely localized in chloroplast membranes. Transgenic expression of PPF1 affected the calcium storage capacities since chloroplasts isolated from PPF1 (+) plants contained high Ca2+ levels while chloroplasts of PPF1 (–) plants contained very low amounts of calcium ion. Using Novikoff human hepatoma cells, we demonstrated that expression of PPF1 leads to a significant inward calcium ion current that was absent in untransformed cells. We conclude that, as a putative calcium ion carrier, PPF1 affects the flowering time of higher plants by modulating Ca2+ storage capacity within chloroplasts.
The flowering of a higher plant is believed to be the result of the sequential action of two groups of genes: the floral meristem identity genes that convert the vegetative apex to a floral meristem and the organ identity genes that direct the formation of various flower parts (Levy and Dean, 1998; Onouchi et al., 2000). Genetic studies of flowering-time mutants subjected to various environmental treatments, such as vernalization and photoperiod, have identified at least four different pathways that control flowering time in Arabidopsis. The somewhat antagonistic floral repression pathway(s) and the autonomous promotion pathway mainly monitor the internal developmental status of the plant while the other two pathways transduce environmental signals. The photoperiodic promotion pathway is only responsible for floral induction under long-day (LD) conditions whereas the vernalization promotion pathway allows flower initiation after an extended period of cold temperature (Levy and Dean, 1998). To date, about 80 genetic loci and more than 40 candidate genes are known to regulate this important process (Aubert et al., 2001; Michaels and Amasino, 2001; Schomburg et al., 2001; Simpson and Dean, 2002; Simpson et al., 1999).
In the G2 genetic line of pea, reproductive growth and apical senescence can be two completely separated processes. Under short-day (SD) growth conditions (9 h of light per 24-h cycle), G2 pea will flower later than its LD (18 h of light per 24-h cycle)-grown counterparts, and the vegetative apical tissue of these SD plants will grow continuously for a very long period of time, with large numbers of flowers and fruit being produced three or four nodes down from the apex. Under LD conditions, G2 pea will flower as early as 4 weeks after germination, and the vegetative apical tissue of these plants ceases growth soon after the initiation of reproduction and undergoes full senescence within another 3–4 weeks as do wild-type plants (Zhu and Davies, 1997; Zhu et al., 1998).
To elucidate the molecular mechanisms underlying the G2 phenotype, we cloned several genes that were expressed specifically in plants growing under the non-senescence inducing SD photoperiod (Li et al., 1998; Zhu et al., 1998). One such gene, PPF1, which was also vigorously upregulated by gibberellin application, shares significant amino acid sequence identity with the Bacillus subtilis vegetative growth-specific factor SP3J, and also with several bacterial inner membrane proteins. A sequence comparison and database search revealed that the PPF1 cDNA shares 74% identity (82% homology) at the deduced amino acid level with the Arabidopsis ALBINO3 (ALB3) gene, which has been reported to be involved in chloroplast biogenesis (Sundberg et al., 1997). Further work suggested that ALB3 might be involved in the post-translational integration of the light-harvesting, chlorophyll-binding protein into thylakoid membranes (Moore et al., 2000). To further assess the function of the PPF1 gene, we generated multiple, independent transgenic Arabidopsis lines expressing the pea PPF1 cDNA driven by the CaMV 35S promoter in both sense and antisense orientations. Genetic and molecular analyses show that the PPF1 gene may negatively regulate flower time through changing subcellular calcium homeostasis.
Phenotypic analysis of transgenic Arabidopsis plants that over- or under-express PPF1 cDNA
Typical phenotypes of PPF1 (+), PPF1 (–), and control plants were shown in Figure 1(a). PPF1 (+) plants (line 5) flower significantly later (77.4 ± 8.1 days post germination (dpg)) than do control plants (52.7 ± 3.3 dpg), whereas PPF1 (–) plants (line 11) display an early flowering phenotype (35.8 ± 4.6 dpg). The differences between these three groups were also reflected when developmental scales were used for comparison. PPF1 (+) plants produced about 25 rosette leaves and the PPF1 (–) plants produced only 8–9 leaves prior to flowering. Under our growth conditions, wild-type Columbia plants usually flower after producing 16–17 rosette leaves (Figure 1b). All the above plants used for phenotypic comparisons were sown at the same time and grown in fully automatic growth chambers under intermediate day-length conditions (12 h light per 24-h cycle). When grown under inductive LD photoperiods the difference in flowering time among PPF1 (+), PPF1 (–), and control plants was not as pronounced (data not shown). However, as will be shown later, when grown under the non-inductive SD photoperiods, greater differences in flowering time were recorded.
PPF1 levels in different plant lines correlate positively to Arabidopsis flowering time
To support the observation that PPF1 functions to delay flowering, we first analyzed the amount of antibody-reactive PPF1 protein present in PPF1 (+), PPF1 (–), and control plants at different growth stages using Western blotting assays. Vigorous expression of the target protein in PPF1 (+) plants coincided with the late-flowering phenotype (Figure 2a). We did not analyze plants that were older than 60 dpg for PPF1 (–) and the control lines as they senesced very rapidly after entering the reproductive stage. Although our antibody recognized both PPF1 and the endogenous ALB3 peptides with comparable affinities (determined in separate experiments using in vitro-expressed PPF1 and ALB3), a single hybridizing band with a calculated Mr of 45 000 was produced in PPF1 (+), PPF1 (–), and wild-type Arabidopsis plants. This is probably due to the fact that the major difference between PPF1 (442 amino acids) and the endogenous ALB3 protein (462 amino acids) is a 20-amino acid insertion in the N-terminal chloroplast transit peptide part of ALB3 that is cleaved upon maturation. Another interesting observation is that many of the more severe antisense lines displayed albino morphology and were not able to go through a full life cycle, a trait that resembles the alb3 mutant (Sundberg et al., 1997).
We also analyzed multiple independent transgenic lines for PPF1 expression, or for the effectiveness of antisensing using SD-grown plants. For all five PPF1 (+) lines that over-expressed the transgene, the transition to flowering, as measured by the number of rosette leaves produced before bolting, was delayed significantly. Similarly, there was a reverse correlation between the time of flowering and the ability of the transgene to suppress the endogenous ALB3 expression (Figure 2b). The regression coefficient obtained using Western blotting data from these plant lines is 0.863.
PPF1 is localized in the chloroplasts of transgenic Arabidopsis plants
We next examined the subcellular location of the PPF1 protein using a GFP fusion construct. When observed under a confocal microscope, trichomes of transgenic Arabidopsis plants carrying the GFP-only construct displayed green fluorescence uniformly throughout the cell. By contrast, in trichomes of transgenic plants expressing the PPF1–GFP construct the green fluorescence was strictly localized in subcellular compartments (Figure 3a). Combining data obtained from both the green and the red channels, we concluded that the fluorescence-emitting small dots are chloroplasts. This conclusion was supported by data obtained from guard cells of the same transgenic Arabidopsis plants as shown in Figure 3(b). Since the chloroplasts were much bigger in this kind of specialized cells, we could see clearly that the green fluorescence was limited to the plastids of plants expressing the PPF1–GFP construct.
Sense or antisense expression of the PPF1 cDNA has a profound impact on the subcellular calcium storage capacity of a plant cell
When the total photosynthetic capacities and subcellular potassium content were measured, we recorded no systematic differences among samples collected from PPF1 (+), PPF1 (–), and wild-type Arabidopsis plant lines as late as 40 dpg (data not shown). However we found substantial differences in the amount of subcellular storage Ca2+ in different plant lines. Guard cells isolated from 30- or 45-dpg PPF1-overexpressing Arabidopsis plants released a significant amount of Ca2+ after addition of 10 µm ionomycin (Figure 4, top panels) in contrast to the lack of significant Ca2+ released from guard cells from PPF1 (–) plants (Figure 4, middle panels). A gradual decay of Ca2+ storage capacity in PPF1 (+) plants was only observed in samples prepared from plants harvested later than 60 dpg (data not shown). Samples taken from 30-dpg wild-type plants released comparable amounts of Ca2+ to that of PPF1 (+) plants, but its Ca2+ storage capacity diminished almost completely over the period from 30 to 45 dpg (Figure 4, bottom panels). We suggest that PPF1 over-expression resulted in a greater and longer-lasting endogenous Ca2+ pool in the chloroplasts.
PPF1 protein expressed in human hepatoma cells can produce a calcium flow
Since PPF1 over-expressers, antisense, and control plants exhibited significantly different subcellular calcium storage capacities, we further examined the possible involvement of PPF1 in calcium flow using Novikoff human hepatoma cells that exhibit no measurable calcium flow in their original state. Indeed, transformed Novikoff cells expressing PPF1 (+) produced a significant inward ion current (Figure 5a) that was not observed in cells transformed with an empty vector (Figure 5b) or in non-transformed control cells (data not shown). Under ramp stimulation conditions, PPF1 (+)-transformed cells exhibited this voltage-dependent inward ion current, first activated at approximately 20 mV and maintained until about approximately 120 mV. The resting potential of transformed cells was about 30 mV more negative than that of control cells, indicating that PPF1 may be an active calcium ion carrier (Figure 5c). This inward current was almost completely suppressed by addition of 100 µm CdCl2 (a specific Ca2+ ion current inhibitor; Lansman et al., 1986) or 40 mm NaN3 (used for blocking the metabolic pathway leading to ATP production), suggesting that the inward current is both Ca2+-specific and energy dependent. Cells that carry an empty vector (Vector-Only) or the antisense PPF1 construct (Vector + PPF1 (–)) produced no calcium ion current (Figure 5d). We also confirmed that the green fluorescence emitted from the GFP-tagged PPF1 protein was localized in plasma membranes of human as well as onion epidermal cells (data not shown).
PPF1 is important for plant growth and development
PPF1 is a developmentally regulated gene first isolated from SD-grown, post-floral G2 pea tissue using cDNA representational difference analysis (cDNA RDA). It was reported that, under this photoperiod, G2 pea displayed a slower reproductive growth rate with an unlimited vegetative potential, accompanied by sustained high-level PPF1 expression (Zhu and Davies, 1997; Zhu et al., 1998). Here, we report two independent lines of evidence that pinpoint the biological importance of PPF1. First, transgenic Arabidopsis plants expressing PPF1 in the sense or antisense orientations showed significant differences in their developmental progression: PPF1 (+) plants flowered very late while PPF1 (–) plants flowered very early (Figure 1). Second, when expressed in human Novikoff hepatoma cells, PPF1 produced an inward calcium ion current that was absent from control cells (Figure 5). Given that chloroplasts in PPF1 (+) plants stored calcium at a significantly higher level compared to PPF1 (–) or control plants (Figure 4), we suggest that PPF1 may function as a chloroplast membrane-localized calcium ion carrier that regulates plant development by releasing different amounts of Ca2+ from its internal pool. These results, together with the previous finding that alb3 mutant could not survive beyond the seedling stage (Sundberg et al., 1997), prompted us to conclude that PPF1 gene may possess an important biological function.
PPF1 overexpression increased subcellular calcium storage capacity of transgenic Arabidopsis plants
Calcium may be the most versatile and universal signaling molecule within all living organisms. It is believed to regulate diverse cellular processes such as heartbeat and information processing in the human brain (Berridge et al., 1998), stomatal opening, and pollen tube re-orientation as well as cell wall thickening (Bush, 1995; Rudd and Franklin-Tong, 1999; Trewavas, 2000). However, apart from a transient elevation of free cytosolic Ca2+ following the fusion of sperm and egg cell in a flowering plant (Digonnet et al., 1997), we know little about its role in flower signal transduction pathways. Experimental results provided in the present study suggested that Arabidopsis flowering might be related to the change of subcellular Ca2+ storage capacity since significant amounts of storage Ca2+ were observed in subcellular compartments of PPF1 (+) Arabidopsis plants as late as 60 dpg (Figure 4). On the other hand, plants that expressed the antisense PPF1 gene exhibited very low Ca2+ storage capacity in subcellular compartments and displayed an early flowering phenotype (Figures 1 and 4). Since these two plant lines differed only in the presence or near-absence of PPF1 (localized to the chloroplast membrane), we suggest that chloroplasts may serve as a major site of subcellular Ca2+ storage in transgenic Arabidopsis plants. The expression and integration of an active calcium ion carrier in chloroplast membrane systems seemed to render the recipient capable of an increased Ca2+ storage capacity. Likewise, antisense suppression of the endogenous ALB3 activity diminished this capacity and caused substantial early Ca2+ release into the cytoplasm (as evidenced in Figure 4). ALB3 was originally proposed to be involved in chloroplast biogenesis or in post-translational integration of light harvesting chlorophyll-binding complex into thylakoid membranes. However experimental data provided in the current work, especially our observation that antisense suppression of ALB3 gene in PPF1 (–) plants did not show any significant decrease in photosynthetic capacity, does not support such a role. Further clarification is needed to explain this discrepancy.
The calcium component in flower-time control
Genetic and biochemical analyses of early- and late-flowering Arabidopsis ecotypes have identified multiple and interwoven regulatory pathways involving many flower promoting or repressing genes (Levy and Dean, 1998; Samach et al., 2000; Simpson and Dean, 2002; Simpson et al., 1999). Most known flowering genes are, however, transcription factors rather than structural genes with bona fide biochemical functions (Amaya et al., 1999; Bewley et al., 2000; Blazquez and Weigel, 2000; Devlin and Kay, 2000; Guo et al., 2001; Kardailsky et al., 1999; Nelson et al., 2000; Sheldon et al., 2000). PPF1 is unique in this respect since it may represent a link between calcium ion transport and the regulation of flowering time in higher plants.
It was previously reported that some Arabidopsis mutants show late-flowering phenotypes under LD growth conditions, but not under SD conditions (Koorneef et al., 1991). Conversely, mutations that block gibberellin biosynthesis abolish flowering under SD conditions but have only minor effects under LD conditions (Wilson et al., 1992). Mutations in the CONSTANS (CO) and GIGANTEA (GI) genes result in late flowering only under LD conditions, whereas flowering locus t (ft) mutants flower late, regardless of the photoperiod, indicating a complexity of developmental regulation (Fowler et al., 1999; Putterill et al., 1995; Ruiz-Garcia et al., 1997). In the present work, over-expression of the SD-specific and gibberellin-inducible PPF1 gene delayed flowering of transgenic plants despite the fact that gibberellic acid (GA) promotes Arabidopsis flowering. We propose that GAs may interact with different receptors or intermediate molecules that are photoperiod specific. During longer photoperiods, GA promotes flowering, presumably through a co-ordinated interaction with cis elements present on the LFY promoter (Blazquez and Weigel, 2000). In shorter photoperiods, GA-activated PPF1 expression may delay flowering by extending the calcium storage function of the chloroplasts.
In conclusion, we find that the Pisum gene, PPF1, affected the flowering time of transgenic Arabidopsis. Plants overexpressing the PPF1 cDNA flower later than controls and plants carrying the antisense construct flower earlier. The PPF1 protein is located in the chloroplast membrane and it is likely a novel calcium ion carrier that may regulate flowering by changing the subcellular Ca2+ storage capacity of the plant cell.
Arabidopsis transformation and seedling growth conditions
The full-length PPF1 gene (Y12168) was amplified from G2 pea by the polymerase chain reaction (PCR) with restriction site-containing primers, and cloned into the binary vector pKF111 containing a glufosinate resistance gene (Ni et al., 1998), either in the sense or antisense orientation. Arabidopsis transformation was carried out using the floral dip method. For the antisense construct, we used a portion of the PPF1 cDNA (from nucleotides 128–1165, with translation initiation site as nucleotides 1–3) that shares over 85% sequence identity with the endogenous ALB3 gene (U89272). Surface-sterilized transgenic and wild-type Columbia seeds were grown at 23°C during the light period and 21°C during the dark period in fully automated growth chambers (Conviron, Canada). Plants were maintained at 9-h (SD), 12-h (intermediate), or 18-h (LD) photoperiods with cool-white fluorescent lamps supplemented by incandescent lamps. Glufosinate ammonium (5 mg l−1; Riededel-de Haen, Germany) was used for transgenic plant selection. Transgenic lines carrying either the PPF1 (+) or PPF1 (–) constructs were selected by semiquantitative RT-PCR assays using the same pair of primers that were PPF1-specific (primer 1: 5′-CGATTCAATCCGTAAGTCAT-3′, primer 2: 5′-GAACTTCCTCCACTGGGAGT-3′) and were verified by Western blotting in T2 generation. Plants homozygous at the transgene locus were tested for single copy segregation before flowering phenotype characterization. Similar growth performances were observed for PPF1 (+) and control plants while PPF1 (–) plants developed slightly slower in terms of leaf production.
Preparation of antibodies against the PPF1 C-terminal peptide
The C-terminal portion of PPF1 (residues 327–442) was cloned into pGEX-4T-1 vector (with GST tag) and transformed into Escherichia coli BL21 cells. The recombinant protein was expressed after IPTG induction and purified using GST affinity resin (Amersham Pharmacia), and the C-terminal PPF1 fragment was cleaved with thrombin (Boehringer Mannheim). Eluant from the resin was further purified by electrophoresis on a 15% SDS–PAGE gel which exhibited a single band migrating at ∼8.5 kDa after staining with Coomassie Brilliant Blue R250. The sequence of the truncated PPFC peptide was confirmed by automated N-terminal peptide sequencing (performed commercially at Peking University) before being used to inoculate rabbits to raise polyclonal antibodies to PPFC.
Apical tissue (excluding the first pair of fully expanded leaves) of various Arabidopsis plants was harvested at noon time and was immediately frozen in liquid nitrogen before being ground into a fine powder using a mortar and pestle. Samples were homogenized in extraction buffer containing 50 mm HEPES-KOH (pH 7.5), 5 mm EDTA, 0.1% BSA, 1 mm PMSF, 2 mm DTT, 1% (w/v) polyvinyl polypyrrolidone (PVP), and 0.25 m sucrose. Cell debris was removed by centrifugation at 10 000 g for 15 min. Immunoblotting of the protein samples transferred to nitrocellulose membranes from a 12% SDS–PAGE gel was performed using 1 : 3000 dilutions of the primary anti-PPFC antibody. In parallel experiments, pre-immune serum was used as the negative control for antibody specificity. Equivalent amounts of total protein, as determined by a protein assay kit (Bio-Rad, USA) were loaded in each of the lanes. Separate experiments using in vitro-expressed PPF1 and its Arabidopsis homolog, ALB3, demonstrated that our antibody recognized both peptides with comparable affinities.
Vector construction and GFP localization in transgenic Arabidopsis
The full-length PPF1 sequence was amplified by PCR and was inserted into the NcoI site of pAVA120 in the same reading frame as the downstream GFP reporter under the control of a CaMV 35S promoter. The stop codon of the PPF1 gene was omitted and a unique NcoI site was added to fuse PPF1 cDNA with GFP. The GFP-only vector contains the GFP coding sequence controlled by the same CaMV 35S promoter. Cellular localization of the GFP reporter was determined using either a confocal microscope or a fluorescence microscope.
Confocal microscopic assays
A confocal laser-scanning microscope (MRC 1024, Bio-Rad, USA) was used to detect GFP fluorescence in both trichomes and guard cells of PPF1–GFP transgenic Arabidopsis leaves. The microscope was equipped with a two-channel scanner and an argon–krypton laser with an excitation wavelength of 488 nm. Twenty sections were acquired by collecting Kalman-filtered scans at 1-µm intervals. Images were recorded as projections using the Confocal Assistant 4.0 software (Bio-Rad, USA). Red and green autofluorescence were emitted by chlorophyll and GFP, respectively.
Potassium, calcium and photosynthetic capacity measurements
Sodium hexanitrocolbaltate staining (Green et al., 1990) and fura-2 fluorescence assays (Minta et al., 1989; Tang et al., 2000) were employed to measure subcellular potassium and calcium concentrations using chloroplasts isolated from various plant lines. The photosynthetic capacities of different Arabidopsis lines were measured using the PAM fluorescence detection method (Schreiber et al., 1995). Intact guard cells were isolated (Schroeder et al., 2001) and incubated first with 50 µm fura-2/AM containing 0.02% Pluronic F-127 in the loading buffer (50 mm KCl, 50 µm CaCl2, 10 mm MES-KOH, pH 5.6) for 1 h to establish cytoplasmic Ca2+-bound fura-2 fluorescence. Ionomycin (final concentration 10 µm) was added to the media following the 1-h incubation to release Ca2+ from subcellular compartments. Calcium fluorescence was viewed with a Nikon Diaphot 200 inverted epifluorescence microscope (Nikon Instruments, USA), and the images were intensified using the MiraCal Imaging system (Life Science Resources, UK). The emitted fluorescence intensities at 510 nm were recorded at 340 and 380 nm excitation wavelengths by Mira-1000TE low-light level CCD camera, and the calcium content was expressed as the ratio of fluorescence measured at 340 nm/380 nm (Tang et al., 2000).
Patch clamp and ion current measurements
The PPF1–GFP coding region was released from the recombinant plasmid by digestion with EcoRI and BamHI and was further cloned into the pLXSN retroviral vector (Clontech, USA), creating a PPF1–GFP fusion driven by the LTR promoter. Novikoff cells (3 × 106 cells ml−1) were transformed with 20 µg ml−1 plasmid DNA using a Micropulser Electroporation Apparatus (Bio-Rad, USA), and were incubated in glass flasks at 37°C for 8 h before use in patch clamp experiments (Hao et al., 2001; Lazrak and Peracchia, 1993). The standard bath solution contained 130 mm NaCl, 2.7 mm KCl, 1.8 mm CaCl2, 2 mm MgCl2, 15 mm glucose, 10 mm HEPES, 15 mm TEA, pH 7.2. The standard pipette solution contained 135 mm KCl, 6 mm NaCl, 0.5 mm MgCl2, 3 mm ATP-Na, 10 mm HEPES, pH 7.2. Where applicable, 100 µm CdCl2 or 40 mm NaN3 (Sigma, USA) were added separately to the bath solution prior to respective measurements. Stimulation and sampling were performed using an amplifier with an A/D–D/A interface (Axon 200B & Digital 1200, Axon Instruments, USA).
We thank J-D. Zhao for help with photosynthetic measurements, T-F Liu for patch clamp study, and Peter Davies for comments and corrections of the manuscript. This work was supported by grants from the Rockefeller Foundation of USA and the Chinese National Natural Science Foundation (Grant 39725002).
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