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First published online October 19, 2007; 10.1104/pp.107.108654 Plant Physiology 145:1692-1702 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The 14-3-3 Proteins µ and
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ABSTRACT |
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 TOP ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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and µ display defects in light sensing and/or response. Mutant plants flower late and exhibit long hypocotyls under red light, with little effect under blue or far-red light. The long hypocotyl phenotype is consistent with a role for 14-3-3 and µ in phytochrome B signaling. Yeast two-hybrid and coimmunoprecipitation assays indicate that 14-3-3 and µ proteins physically interact with CONSTANS, a central regulator of the photoperiod pathway. Together, these data indicate a potential role for specific 14-3-3 isoforms in affecting photoperiodic flowering via interaction with CONSTANS, possibly as integrators of light signals sensed through the phytochrome system.
). 14-3-3 proteins may also impart their control by binding nonphosphorylated proteins (Masters et al., 1999), cruciform DNA (Todd et al., 1998), and can bind protein partners as a monomer (Woodcock et al., 2003).
However, the most common means by which 14-3-3 proteins influence signaling events entails the binding of a phosphorylated protein target by a 14-3-3 dimer (Ferl, 1996), rendering the 14-3-3 proteins as candidate factors in the regulation of signal transduction pathways. Kinases contribute to the phosphorylation status of a target protein, which is then negated or promoted in the progression through a signal transduction pathway via association with 14-3-3s. The phosphorylation events in signal transduction pathways present ideal opportunities for 14-3-3 participation, as the binding of 14-3-3 proteins to phosphorylated proteins completes the signal-induced change in activity (Sehnke et al., 2002).
Participation of 14-3-3 proteins within signal transduction pathways has been extensively examined in animal systems, where 14-3-3 proteins occupy a central role. However, research in plant signal transduction has largely characterized 14-3-3 proteins as components of the terminal ends of pathways. Some examples of 14-3-3 protein participation in the termini of signal transduction pathways include 14-3-3 activation of the plasma membrane H(+)-ATPase (Olsson et al., 1998; Fuglsang et al., 1999; Svennelid et al., 1999), the regulation of the metabolic enzymes nitrate reductase and Suc phosphate synthase (Bachmann et al., 1996; Toroser et al., 1998), association with the transcription machinery (Pan et al., 1999; Carrasco et al., 2006), and interaction with the plasma membrane H(+)-ATPase in guard cells in a blue-light-dependent manner (Kinoshita and Shimazaki, 1999). On the input side of signaling pathways 14-3-3 proteins interact with transmembrane receptor-like kinases (Rienties et al., 2005) and also interact with the phototropin blue light receptor (Kinoshita et al., 2003; Ueno et al., 2005).
Isoform-specific function of 14-3-3 proteins has been described in animal signal transduction systems. Similar trends may be expected in plants. However, actions of specific 14-3-3 isoforms in plant signal transduction pathways have yet to be phenotypically elucidated, possibly because the large size of the 14-3-3 gene family in most plants presents potential for functional redundancy. Thus, there is a need to understand the roles of specific 14-3-3 isoforms in plants and to address issues of functional redundancy. Here well-characterized T-DNA insertion mutants allow a gene-specific description of isoform action and contribution to discrete signaling processes.
One fundamental plant signal process that might be predicted to be impacted by 14-3-3s is the transition between vegetative and reproductive growth, which is a tightly regulated process governed by at least five different signaling inputs: photoperiod, light quantity, light quality, vernalization, and nutrient/water availability (Cerdan and Chory, 2003; for review, see Levy and Dean, 1998; Putterill et al., 2004; Amasino, 2005). Precise coordination of flowering time promotes a higher likelihood of cross pollination and seasonal coordination of fruit set. The pathways that regulate this developmental transition are rife with biochemical events and modifications that are akin to demonstrated 14-3-3 activities in animals. Phytochromes possess kinase activity and are regulated by phosphorylation events (for review, see Kim et al., 2005), ubiquitination (Clough et al., 1999), compulsory localization to the nucleus (Yamaguchi et al., 1999; Huq et al., 2003; Valverde et al., 2004), and the binding to G-box elements (Chattopadhyay et al., 1998; Martinez-Garcia et al., 2000). CONSTANS (CO) is a transcriptional regulator that is also likely regulated by phosphorylation and nuclear localization (Valverde et al., 2004). All of these activities are consistent with well-described activities associated with 14-3-3 proteins (Lu et al., 1992; Igarashi et al., 2001) and present logical targets for assessment of 14-3-3 regulation of specific plant processes.
In this study, Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant lines for 14-3-3 (14-3-3-1 and 14-3-3-2) and 14-3-3 µ (14-3-3µ-1) have been characterized at the protein production level and then assayed for phenotypic variations from wild type (Columbia-0 ecotype). The 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 alleles present alterations in the timing of flowering and hypocotyl growth inhibition during early development. To identify the nodes where 14-3-3 µ and influence the signaling network, 14-3-3 µ and were tested for interactions with several phytochrome, clock, and photoperiod pathway components using functional and biochemical assays. The combination of 14-3-3 mutant phenotypic assays and 14-3-3 protein interaction data indicate a likely role for 14-3-3 and µ in phytochrome-mediated light input and physical association with members of the photoperiod pathway.
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RESULTS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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Homozygous T-DNA insertion mutants containing lesions in 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 were identified in the Salk T-DNA insertion mutant collection using PCR. 14-3-3-1 contains a T-DNA insertion within the third exon of the 14-3-3 gene and 14-3-3-2 within the second intron (Fig. 1A
), while 14-3-3µ-1 contains a T-DNA insertion located directly 5' to the coding region of the 14-3-3 µ gene (Fig. 1B). A second allele for the disruption of the 14-3-3 µ gene was not available. To have an adequate negative control for phenotypic assays utilizing 14-3-3µ-1, a genomic copy of the 14-3-3µ gene was introduced to the mutant and assayed in parallel with the genotype bearing the disrupted gene. This genetic complementation line contains the native 14-3-3 µ gene with the native 14-3-3 µ promoter, and was integrated into the 14-3-3µ-1 mutant genome using Agrobacterium-mediated transformation ("Materials and Methods"). The 14-3-3µ-1 complementation line was named Res-mu.
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). The anti-14-3-3 immunoblot confirmed that 14-3-3-1 and 14-3-3-2 do not express any detectable 14-3-3 protein (Fig. 1A), while wild-type, Res-mu, and 14-3-3µ-1 lines expressed comparable levels of 14-3-3 protein (Fig. 1B). The 14-3-3 µ immunoblot confirmed that 14-3-3-1 expressed wild-type levels of 14-3-3 µ (Fig. 1A), while 14-3-3µ-1 produced only a fraction of wild-type 14-3-3 µ protein levels (Fig. 1B). Res-mu protein extracts contained concentrations of 14-3-3 µ protein at higher levels than 14-3-3µ-1, but were not quite equal to wild-type protein extracts (Fig. 1B). Separate immunoblots of the same membranes using an antibody specific for 14-3-3 and 14-3-3 µ (Fig. 1, A and B) demonstrated that the loss in expression of one 14-3-3 isoform does not affect the expression of the other 14-3-3 isoform.
It is important to note that the 14-3-3µ-1 T-DNA insertion does not completely disrupt the accumulation of 14-3-3 µ protein (as does 14-3-3-1 and 14-3-3-2 for the 14-3-3 protein), as equal amounts of protein were loaded for the assay of 14-3-3µ-1 protein levels. Twelve micrograms of wild-type, Res-mu, and 14-3-3µ-1 protein extracts were loaded onto the lanes of the SDS-PAGE gel. Further, lanes were skipped to avoid protein carryover into adjacent lanes.
The difference in the 14-3-3 T-DNA insertion mutant's expression levels of 14-3-3 protein and 14-3-3µ-1 of 14-3-3 µ protein may be due to the differences in T-DNA insertion localization. The 14-3-3 T-DNA insertion mutants have a T-DNA in the coding region of the 14-3-3 gene (Fig. 1A). The T-DNA insertions within the 14-3-3 T-DNA insertion mutants cause a disruption of the protein sequence, possibly resulting in a truncated protein that is quickly degraded. However, 14-3-3µ-1 has a T-DNA insertion within the promoter region of the 14-3-3 µ gene (Fig. 1B). Location of the 14-3-3µ-1 T-DNA allows for the expression of wild-type 14-3-3 µ, although expression occurs at a reduced level resulting from disruption of likely 14-3-3 µ regulatory sequences. This is an important consideration, as the results to follow do indicate some overlapping function.
14-3-3 Mutants Exhibit a Delay in Flowering
Disruption of either the 14-3-3 or µ locus results in a delay of flowering (Fig. 2, A and C
). The 14-3-3 T-DNA insertion mutant lines and wild-type plants were grown under long-day (16-h day, 8-h night) conditions. Emergence of the first flower varied by 3 d between 14-3-3-1/14-3-3-2 and wild type and 6 d between 14-3-3µ-1 and wild type (Fig. 2C). Flowering experiments were repeated for multiple generations with comparable results. t tests confirmed differences in the average day of flowering between wild type and 14-3-3-1, 14-3-3-2, or 14-3-3µ-1 were significant (
< 0.0001, 0.0002, and 0.0001, respectively). Flower emergence was defined as the first appearance of petals.
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-1 and 14-3-3-2 mutants presented a one-leaf average increase in leaf number and 14-3-3µ-1 a two-leaf average increase in comparison to wild type (Fig. 2E). Leaf number experiments were repeated for several generations with comparable results. t tests confirmed differences in leaf number between wild type and 14-3-3-1, 14-3-3-2, or 14-3-3µ-1 were significant ( = 0.002, 0.023, and 0.0007, respectively). The increased average leaf number results indicated that 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 exhibited a delay in the transition to flowering rather than an overall delay in development. To ensure that phenotypes observed were related to the T-DNA insertion in the 14-3-3µ gene and were not an artifact of other genomic modifications, the Res-mu line was developed as a specific negative control for the 14-3-3µ-1 line. In a separate flowering timing experiment, timing of Res-mu transition to flowering was assayed in comparison to that of 14-3-3µ-1 (Fig. 2, B and D). Res-mu exhibited a 5 d decrease in time to flowering in comparison to 14-3-3µ-1 (Fig. 2D), demonstrating a nearly complete reversal in the delay caused by the original 14-3-3µ-1 T-DNA insertion. The Res-mu line also reversed the increase in number of leaves at the time of flowering normally exhibited by 14-3-3µ-1 (Fig. 2F), indicating that the delay in flowering was due to a delay in the floral transition.
14-3-3 T-DNA insertion mutant flowering was monitored under short-day conditions to test if 14-3-3 proteins were associated with the photoperiod pathway and were not simply negative regulators of the floral transition (Fig. 3
). Mutants for photoperiod regulating genes, such as CO and Gigantea (Gi) flower later during long-day conditions, yet flower at the same time as wild type in short days (Redei, 1962; Koornneef et al., 1991). Wild type, co mutants (co-1; Redei, 1962; Koornneef et al., 1991), and the 14-3-3 T-DNA insertion mutants were assayed for timing to flowering and leaf number at the time point of flowering, under short-day (8 h light/16 h dark) conditions (Fig. 3). The co and 14-3-3 T-DNA insertion mutants all exhibited flowering timing that was slightly sooner in comparison to wild type (Fig. 3A), which is consistent with previous observations of co grown under short-day conditions (Putterill et al., 1995). However, co and the 14-3-3 T-DNA insertion mutants produced an average number of leaves at the time point of flowering that was comparable to wild type. A t test confirmed that the number of leaves at the time point of flowering was not significantly different between wild type and co, 14-3-3-1, 14-3-3-2, or 14-3-3µ-1 ( = 0.5207, 0.2052, 0.4102, and 0.7177, respectively).
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Red, blue, and far-red wavebands play discrete roles in the flowering transition following activation of their cognate receptors (Valverde et al., 2004). An effective way to test the possible mechanisms accountable for deficiencies in light input to the photoperiod pathway is to compare early photomorphogenic responses in 14-3-3 T-DNA insertion mutants and wild-type seedlings in response to narrow-bandwidth red, blue, or far-red light (Neff and Chory, 1998). Aberrant seedling development under a specific wavelength of light may indicate participation in a particular photosensory pathway. The phytochromes are receptors of red and far-red light (also blue and UV-A) whereas phototropins and cryptochromes are the dominant blue and UV light receptors (Christie and Briggs, 2001; Lin, 2002). Since phytochromes and cryptochromes are the dominant receptors controlling hypocotyl growth inhibition, tests of growth inhibition under specific wavelengths may indicate 14-3-3 participation in a specific light-sensing/response pathway. Decreased hypocotyl growth inhibition (resulting in a longer hypocotyl after days in light) is observed when a mutant is less sensitive to the particular wavelength of light being tested (Parks et al., 2001). Conversely, mutants hypersensitive to a particular wavelength will display increased hypocotyl growth inhibition under the particular wavelength of light being tested.
Wild-type, Res-mu, 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 were grown under narrow-bandwidth red, blue, or far-red light wavelengths for 4 d and then measured to assess hypocotyl growth inhibition. 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 all displayed reduced hypocotyl growth inhibition under 1 and 10 µmol m–2 s–1 red light relative to dark-grown seedlings (Fig. 4
). 14-3-3-1 and 14-3-3-2 displayed a 22% and 32% longer hypocotyl than wild type, respectively, when grown under 1 µmol m–2 s–1 of red light and a 19% and 36% longer hypocotyl, respectively, when grown under 10 µmol m–2 s–1 of red light. 14-3-3µ-1 and Res-mu exhibited a 30% and 17% longer hypocotyl, respectively, in comparison to wild type at 1 µmol m–2 s–1 of red light and a 48% and 14% longer hypocotyl, respectively, when grown under 10 µmol m–2 s–1 of red light. Res-mu inhibition of hypocotyl elongation was more comparable to that of wild type than to 14-3-3µ-1 when grown under red light, demonstrating that the difference in hypocotyl elongation inhibition between wild type and 14-3-3µ-1 can be attributed to 14-3-3µ mutation and not second-site T-DNA insertion effects. The decrease in growth inhibition of the 14-3-3 T-DNA insertion mutants under low-intensity, narrow-bandwidth red light indicates that the 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 are less sensitive to red light, placing 14-3-3 and µ as likely components in the phytochrome input or response pathway. The 14-3-3 T-DNA mutants did not display significant defects in hypocotyl growth inhibition when grown under blue light or far-red light (data not shown), suggesting that the activity is related to phytochrome B (phyB) or other phytochromes, acting alone or in combination.
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-1 Displays a Directional Growth Phenotype under Red Light
Wild-type Arabidopsis exhibits a random directional growth pattern when grown under red light (Liscum and Hangarter, 1993). The random directional growth habit under red light phenomenon is not fully understood. However, phyA, phyB, and phyA phyB mutants exhibit a less randomized, vertical directional growth habit under monochromatic red light (Liscum and Hangarter, 1993; Poppe et al., 1996; Robson and Smith, 1996).
Since 14-3-3 mutants and phytochrome mutants present defects in flowering and exhibit phenotypes in hypocotyl growth inhibition, it was of interest to note any deviations in vertical growth habit associated with 14-3-3 T-DNA insertion mutants or mutants of other photoperiod regulatory proteins. Common phenotypes may allow further resolution of how 14-3-3 proteins associate with light sensing and the flowering mechanism. Wild-type randomization of growth direction in red light is reduced in phyA and phyB mutants (Poppe et al., 1996), and has not been reported in seedling growth assays for phyC, phyD, or phyE mutants (Franklin et al., 2003; Monte et al., 2003; Balasubramanian et al., 2006). Therefore, such patterns will allow discrimination of phyA and phyB effects apart from those of other phytochromes that exhibit weak stem elongation defects. Arabidopsis seeds were germinated and grown under narrow-bandwidth red light (660 nm) at 10 µmol m–2 s–1 for 4 d (Fig. 5
). In agreement with previously published findings (Liscum and Hangarter, 1993), wild-type Arabidopsis exhibited a more randomized directional growth relative to gravity under monochromatic red light, whereas phyB (Salk_022035 and Salk_069700; "Materials and Methods") mutants exhibited a more vertical tendency. GI, another protein central to flowering and other phytochrome responses (Huq et al., 2000) was also studied. The gi (gi-2) mutants did not grow as vertically under narrow-bandwidth red as phyB, but gi did exhibit a more vertical directional growth habit than wild type. The co mutants exhibited a wild-type growth pattern. Consistent with sensitivity to red light in hypocotyl growth inhibition assays, the 14-3-3-1 and 14-3-3-2 mutants also exhibited a phyB-like vertical growth habit, further supporting a 14-3-3 functional association with the PHYB light-sensing pathway.
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-1/14-3-3-2 and 14-3-3µ-1. The 14-3-3µ-1 plants were more similar to wild type. However, 14-3-3-1 and 14-3-3-2 exhibited a phenotype similar to phyB. These data indicated that 14-3-3-1 phenocopies phyB and photoperiod-associated mutants in another early developmental response to light.
It should be noted that the timing of flowering in plants is influenced by a number of different environmental and signaling factors. Direct influences of environment on 14-3-3s (such as differential temperature and light quality) can be eliminated as the cause of the 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 delay in flowering phenotype, since the wild type and the T-DNA mutants were grown in identical conditions. The more likely and immediately tractable solution involves association of 14-3-3 proteins with the light sensory inputs to the photoperiod pathway.
14-3-3 Proteins Interact with CO
The influence of 14-3-3 and µ in flowering and early development indicated functional interaction with PHYB, possibly as a direct mechanistic link between PHYB and the photoperiod regulatory systems, including those that may include alterations in the circadian oscillator. To test this hypothesis, yeast two-hybrid assays were utilized as an assay for interactions between 14-3-3 or µ with proteins for the circadian oscillator and photoperiod pathway (Fig. 6
). Two methods were utilized to test interaction between 14-3-3 proteins and candidate proteins; orthonitrophenyl-β-galactoside (ONPG) assays were utilized to measure the β-galactosidase production resulting from protein-protein interactions, while growth assays relied on protein-protein interactions to activate auxotrophic markers required for yeast growth. Direct interaction between 14-3-3 isoforms and specific regulatory proteins would provide positive evidence with a 14-3-3 linkage with the photoperiod pathway.
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with GI, ZEITLUPE (ZTL), PHYTOCHROME INTERACTING FACTOR3 (PIF3), TIMING OF CAB EXPRESSION1 (TOC1), CO, and PHYB. Heterodimer formation was also assayed between 14-3-3 µ and , as a positive control (Wu et al., 1997), while empty vectors were tested against the two 14-3-3 isoforms to control for 14-3-3 self activation. To test 14-3-3 interactions with photoperiod-associated proteins, yeast was transformed with one DNA binding domain vector (pDEST32), containing a 14-3-3 or µ gene and one activation domain vector (pDEST22), containing a photoperiod-pathway gene. Heterodimer formation was tested with yeast containing 14-3-3 in pDEST32 and 14-3-3 µ in pDEST22. Yeast strains used as a negative control contained either 14-3-3 or µ in pDEST32 and an empty pDEST22.
Yeast growth assays were used to test for the ability of the yeast to grow on media lacking uracil (Ura) and His. Interaction was indicated by growth on –Ura/–His dropout media. Of the many central oscillator and photoperiod pathway-associated proteins tested, interaction was only observed between CO with both 14-3-3 and µ (Fig. 6A). Yeast lines containing 14-3-3 fused to the activation domain and 14-3-3 µ fused to the DNA-binding domain also resulted in growth, presumably from the formation of 14-3-3 protein dimers.
The growth assay results were verified using ONPG enzymatic assays in which β-galactosidase activity was measured in Miller units (Miller, 1972). Results from the ONPG assays revealed that yeast expressing CO fused to the activation domain with either 14-3-3 fused to the DNA-binding domain exhibited β-galactosidase activity at higher levels than background (Fig. 6B). The yeast line expressing 14-3-3 fused to the DNA-binding domain and 14-3-3 µ fused to the activation domain also resulted in β-galactosidase levels higher than background, providing a positive control from known interaction between 14-3-3 proteins as dimers. No other yeast lines exhibited β-galactosidase activity higher than background.
Coimmunoprecipitation from cellular extracts also demonstrates a CO-14-3-3 interaction. Leaf extracts from plants overexpressing CO (similar to published tests where overexpressing plants were employed, including Valverde et al., 2004) were immunoprecipitated with anti-CO antibodies and the precipitated proteins were probed with a 14-3-3 monoclonal antibody (Sehnke et al., 2006). Cross-reacting 14-3-3 protein was detected in the anti-CO precipitation lanes but not in lanes from extracts mock precipitated without the primary anti-CO antibody (Fig. 6C).
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DISCUSSION |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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The 14-3-3 T-DNA insertion mutants, 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 were characterized for their specific 14-3-3 protein expression and then assayed for phenotypes. Conspicuous developmental deficiencies were identified in early photomorphogenic development and flowering. The best interpretation of these data suggests a role for 14-3-3s in red light signaling, most likely through phyB. Hypocotyl measurement revealed that 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 exhibit a decrease in hypocotyl growth inhibition under low fluence rate red light, but not blue or far-red light. This hyposensitivity is best interpreted as a defect in phytochrome input. These observed defects are corrected by a native-promoter-driven complementation construct, Res-mu, which almost completely restored normal hypocotyl growth inhibition under low fluence rate red light (Fig. 4). The partial complementation from Res-mu is most likely due to the expression level of 14-3-3 µ protein in the Res-mu line (Fig. 1B), as Res-mu clearly expresses more 14-3-3 µ protein than 14-3-3-1, but does not express 14-3-3 µ protein to the same level as wild type. The correlation between 14-3-3 µ protein level and hypocotyl growth inhibition is strong.
Directional growth in monochromatic red light is also phytochrome dependent, and 14-3-3-1 and 14-3-3-2, but not 14-3-3µ-1 exhibit directional growth, indicating a decreased sensitivity to red light. Wild-type seedlings grown on vertical agar plates under red light adopt a more randomized growth pattern relative to gravity, whereas phyB grows toward the gravitational vector. These findings again place these 14-3-3 mutants in functional association with phyB. These observations are important because while phyC mutants possess similar hypocotyl defects under some conditions, previous reports do not describe defects in red light orientation in phyC or other phytochrome loci. Therefore, the specific hyposensitivity to red light in growth inhibition and directional growth assays places these 14-3-3 isoforms into the phyB pathway. Formal tests of epistasis will more completely delineate these associations.
If 14-3-3 isforms are truly associated with phyB input, effects in photoperiodic flowering would be expected since phyB mutants exhibit deviations in flowering behavior, as phyB sets and conditions the circadian oscillator as well as regulates the posttranslational stability and nuclear localization of CO (Valverde et al., 2004). Consistent with this expectation the 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 lines exhibited a small, yet significant and reproducible delay in the transition to flowering under long-day growth conditions. The 14-3-3-1 and 14-3-3-2 lines had an average of 3 d and 14-3-3µ-1 a 6 d delay in the transition to flowering, relative to wild-type plants. To determine if the flowering phenotype could be attributed to overall slower development of 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 or delay in the transition to flowering, leaves were counted at the time of first flower emergence. If 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 exhibited a phenotype of overall slower development, leaf number at the time of flowering would be the same as found on wild-type plants. If the flowering phenotype could be attributed to a delay in the time to transition to flowering, the 14-3-3 T-DNA insertion mutants would exhibit an increase in the number of leaves at the time of first flower emergence. An increase in leaf number at the point of flowering would indicate that the mutants were dedicated to vegetative growth for a longer period of time before the transition to flowering occurs. The 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 mutants did indeed yield an increased number of leaves at the time of flowering (Fig. 2B), indicating a delay in the transition to flowering. It is also important to note that the 14-3-3 µ protein is reduced but still detectable in the 14-3-3µ-1 T-DNA insertion mutant (Fig. 1) and that a delay in transition to flowering is nonetheless observed. The complementation construction, Res-mu, flowered normally, indicating that differences in flowering observed between wild type and 14-3-3µ-1 were due to the disruption of 14-3-3 µ expression and not another T-DNA insertion event within the 14-3-3µ-1 genome.
While hypocotyl growth assays and measurements of flowering time all demonstrate a likely role for these 14-3-3 isoforms in phytochrome signaling, tests of 14-3-3 interaction with specific clock and photoperiod components would indicate the potential node(s) of 14-3-3s regulation of red light sensing and downstream components. Yeast two-hybrid interactions were used to directly test physical association between 14-3-3 isoforms and proteins demonstrated to play pivotal roles in light sensing and photoperiodic flowering, and circadian periodicity. 14-3-3 interaction was only observed with CO, a central element of the photoperiod pathway. CO has a CO, CO-like, and CCT (timing of CAB1) domain (Putterill et al., 1995), a conserved C-terminal protein-protein interaction domain shared with a suite of other proteins including TOC1 and CO-LIKE proteins (Strayer et al., 2000; Robson et al., 2001). In this study interaction was not observed between 14-3-3 proteins and TOC1, indicating potential specificity for sequences within the CO protein outside of the CCT domain. An in vivo association between 14-3-3 and CO in planta is supported by co-IP from leaves of 14-3-3s with anti-CO antibodies. Modification and/or relocalization of target proteins are phenomena associated both with 14-3-3 interactions and with critical regulatory facets of CO's role in flowering (Valverde et al., 2004). Ongoing studies will test the effect of 14-3-3 lesions on CO stability and nuclear transport under appropriate spectral conditions and photoperiod.
The compilation of 14-3-3-1, 14-3-3-2, and 14-3-3µ-1 phenotypic data shows that 14-3-3 and µ proteins are functionally associated with the PHYB pathway in early development. On the other hand, the delay in transition to flowering displayed by 14-3-3-1 and 14-3-3µ-1 is contrary to the phyB mutant flowering phenotype, which is typified by an accelerated transition to flowering (Goto et al., 1991; Reed et al., 1993). While seemingly difficult to reconcile at first consideration, these data are completely consistent with potential roles of 14-3-3 proteins. The 14-3-3 proteins regulate signals by facilitating protein-client interactions. It is entirely conceivable that the 14-3-3 may spur the activity of a negative phytochrome regulator during early development in the hypocotyl yet excite a positive regulator in the mature leaf, or that 14-3-3 proteins may be acting on targets downstream of PHYB, a hypothesis allowed by the lack of direct interaction between 14-3-3 isoforms and PHYB in the current yeast two-hybrid assays.
The data presented here make a compelling suggestion that 14-3-3s participate in the mechanisms that govern light sensing and photoperiodic flowering control by interacting with CO and regulation some aspects of red light integration into the photoperiod pathway. The physiology of the 14-3-3 mutants indicates associations with phyB and photoperiod. The biochemistry indicates interaction with CO. Thus the involvement of 14-3-3s is likely to be complex. Indeed, the phenotypic effects of these individual 14-3-3 mutations, though significant, are relatively small, further suggesting the likelihood that other factors contribute to this regulatory interaction, including possibly other members of the Arabidopsis 14-3-3 family. Combining these characterized 14-3-3 mutations with mutations in the photoperiod pathway should further elucidate the potential mechanisms of 14-3-3 involvement in the pathway. Experiments characterizing additional downstream effects of CO activity should further elucidate the potential impact points affected by this interaction.
This study addresses fundamental questions in 14-3-3 plant biology, as these are the first insertion mutations described for 14-3-3s that are well characterized at the protein expression level and that display developmental or morphological phenotypes. Therefore it is now possible to use genetics to address the functional overlap and specificity in Arabidopsis 14-3-3s. Certainly 14-3-3 and µ share partial redundancy for the phenotypes described, but do not share a full overlap of function in terms of phenotypic intensity. Pyramiding mutations into many family members may reveal more intense phenotypes as functional redundancy is overcome. Further, the interactions with CO and the roles of 14-3-3 association with PHYB in red light sensing and signaling must be further examined to test direct relationships among 14-3-3s, CO, and light input mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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Seed stocks for 14-3-3-1 (Salk_014690) and 14-3-3µ-1 (Salk_004455) were obtained from the Arabidopsis Biological Resource Center (ABRC). Homozygous T-DNA mutant lines were identified using PCR. A left border T-DNA-specific primer (LBb1) and two, T-DNA flanking, 14-3-3 gene-specific primers were used to identify lines that were homozygous for T-DNA insertion into the respective 14-3-3 genes.
Development of the Res-mu Genetic Complementation Line
The 14-3-3 µ gene with the native promoter and introns was integrated into the genome of 14-3-3µ-1 using the pKGW vector (Karimi et al., 2002). The primers 5'-ggggacaagtttgtacaaaaaagcaggctcctgcagcttcgtgacccat-3' and 5'-ggggaccactttgtacaagaaagctgggtcggatagatagtaagacga-3' were used to PCR amplify the 14-3-3 µ gene from a genomic DNA extract of wild-type Arabidopsis (Arabidopsis thaliana) of the ecotype Columbia-0. The PCR products were then transferred into the Gateway pDONR221 vector using Gateway BP Clonase II (Invitrogen) and subsequently transferred into pKGW from pDONR221 using the Gateway LR Clonase II recombination reaction. The pKGW vector harboring the 14-3-3 µ gene was then transformed into Agrobacterium and introduced into 14-3-3µ-1 plants by the floral-dip method (Clough and Bent, 1998).
Protein Assays of the 14-3-3 T-DNA Mutant Lines
To test protein levels in T-DNA insertion lines, protein extracts were assayed using polyclonal antibodies directed against specific 14-3-3 isoforms. This assay was important because the µ allele contained an insertion into the 5' promoter region. Protein extracts were collected from 14-3-3 mutant and wild-type plants that were grown for 3 weeks under constant light conditions (60–70 µmol m–2 s–1; two Cool White Sylvania bulbs and two Gro-Lux bulbs). Protein extractions for testing 14-3-3 protein levels of the 14-3-3-1 and 14-3-3-2 were performed by grinding one or two leaves in SDS/β-mercaptoethanol (BME) extraction buffer (1 mM Tris-HCl, 0.6% SDS, 20% glycerol, 5% BME, 0.5% bromphenol blue), which was then placed in boiling water for 10 min. The protein extracts were then centrifuged at 20,800g for 10 min to clear insoluble debris.
A HEPES protein extraction buffer (20 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 0.1% Tween 20, and 0.2% protease inhibitors [product no. P9599; Sigma-Aldrich]) was used to extract protein from 14-3-3µ-1, the 14-3-3µ-1 complementation mutant (Res-mu), and wild-type leaf tissue. Approximately 100 mg of leaf tissue was extracted from 2- to 3-week-old plants, frozen in liquid nitrogen, ground with a mortar and pestle, and placed in 500 µL of HEPES protein extraction buffer. The protein extraction solution was then centrifuged at 10,000 rpm for 5 min to separate insoluble material. The supernatant was then extracted and quantified with a Nanodrop ND-1000 (Nanodrop) spectrophotometer. Proteins were then diluted to 1.2 mg/mL with SDS/BME extraction buffer.
A total of 20 µL of each protein sample was fractionated by SDS-PAGE. The proteins from the SDS-PAGE gel were transferred to a nitrocellulose membrane and the membrane was treated overnight with blocking buffer (5% nonfat milk, 0.8% NaCl, 0.02% KCl, 0.1% Na2HPO4, 0.02% KH2PO4). The membranes were incubated with approximately 1:2,000 concentration of antibodies (anti-14-3-3 or anti-14-3-3 µ) in blocking buffer, followed by sequential incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody, then Pierce Supersignal West Pico Chemiluminescent Substrate (product no. 34080; Pierce Biochemical). Pierce western blot stripping buffer (product no. 21059; Pierce Biochemical) was used to strip antibodies from the immunoblots before probing with a separate primary antibody. Chemiluminescent signal was visualized using a Kodak Gel Logic 440 imager.
Flowering Time Assays
Seeds of the 14-3-3 T-DNA insertion mutant lines and wild-type Arabidopsis were planted on wet Fafard Fine-germinating mix soil and placed in 4°C in dark conditions for 3 d. The plants were grown under 60 to 70 µmol m–2 s–1 of light (two Cool White Sylvania bulbs and two Gro-Lux bulbs) with 16 h day, 8 h night photoperiod at 22°C to 24°C. Data were collected from replicates of 18 developmentally uniform 14-3-3-1, 14-3-3-2, 14-3-3µ-1, Res-mu, and wild-type plants. Flowering was scored as the first appearance of petals. Flowering number was scored as the total number of rosette leaves at the time of flowering. The number of leaves did not include the cauline leaves.
Hypocotyl Growth Inhibition and Directional Growth Assays
Arabidopsis seeds were sterilized as previously described (Paul et al., 2001). Thirty to 40 seeds of each line were planted onto minimal media, which contained 1 mM MgCl2, 1 mM CaCl2, and 0.8% agar. After planting, the seed-containing plates were wrapped in foil and incubated at 4°C for 48 h. The seeds were then exposed to white light for 1 h and placed under light emitting diode light apparatuses. Treatments included irradiation with specific fluence rates of red or far-red light (660 and 630 nm, Quantum Devices) or blue light (470 nm, Ledtronics Inc.; and a custom design used by Folta et al., 2005). The spectra and composition of the light sources used are presented online at http://www.arabidopsisthaliana.com/lightsources. End point growth inhibition was evaluated after 96 h. Hypocotyl lengths were compared as the percentage of hypocotyl length of 4-d-old dark-grown seedlings relative to red-grown seedlings such that the assay measures specifically the response to light and not intrinsic length. Digital images were immediately captured using an Epson Perfection 3170 Photo scanner for further analysis. Growth inhibition was assessed by measuring hypocotyls with the University of Texas Health Science Center San Antonio Dental School Image Tool software (http://ddsdx.uthscsa.edu/dig/itdesc.html) and compared to elongation in darkness. The University of Texas Health Science Center San Antonio Dental School Image Tool was also utilized for measuring the angle of deviation from vertical directional growth. The measurements collected were standardized and processed in Microsoft Excel. Directional growth diagrams were constructed by opening pictures of red light-grown plants in Microsoft PowerPoint and tracing lines over each individual plant in the picture, then centering the lines upon one point.
Characterization of the PHYB T-DNA Insertion Mutant Lines
The putative PHYB T-DNA insertion mutant lines, Salk_022035 and Salk_069700, were also obtained from ABRC and PCR screened for the homozygous lines, as described above for the 14-3-3 T-DNA insertion mutant lines. However, the seeds obtained from ABRC were first grown under 1 µmol m–2 s–1 of red light for 4 d (as described above for hypocotyl growth inhibition assays) and the plants with longer hypocotyls were selected for PCR screening. One-hundred percent of the red light-grown Salk_022035 and Salk_069700 plants with longer hypocotyls were found to be homozygous for the respective T-DNA insertion.
Yeast Two-Hybrid Yeast Strains
Yeast (Saccharomyces cerevisiae) were transformed using the Invitrogen ProQuest two-hybrid system with Gateway Technology (Invitrogen). Leaf and flower cDNA was used as the template to PCR amplify the genes of each protein studied. The primer sets used for PCR are as follows (uppercase distinguishing the Gateway cloning recombination region): 14-3-3 , 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatgtcttctgattcgtcc-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCtcactgcgaaggtggtgg-3'; 14-3-3 µ, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatgggttctggaaaagagc-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCtcactctgcatcgtctcc-3'; PIF3, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatgcctctgtttgagctttt-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCtcacgacgatccacaaaac-3'; GI, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatggctagttcatcttcatc-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCttattgggacaaggatatagt-3'; CO, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatgttgaaacaagagagtaa-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCtcagaatgaaggaacaatccca-3'; ZTL, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatggagtgggacagtggt-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCttacgtgagatagctcgct-3'; TOC1, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatggatttgaacggtgagtg-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCtcaagttcccaaagcatcat-3; and PHYB, 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCatggtttccggagtcggg-3'/5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCctaatatggcatcatcagcatc-3'. The PCR products were then transferred into the Gateway pDONR221 vector using Gateway BP Clonase II. The genes of each pDONR221 plasmid were sequenced to confirm proper recombination. The GI, ZTL, PIF3, TOC1, CO, and PHYB genes of pDONR221 were transferred to the pDEST22 destination vector and 14-3-3 and µ to pDEST32 using the Gateway LR Clonase II recombination reaction. The MaV203 yeast strain was transformed with a DNA-binding domain destination vector (pDEST32) and an activation domain destination vector (pDEST22) for each yeast two-hybrid interaction study.
Yeast Two-Hybrid β-Galactosidase Assays
Yeast two-hybrid β-galactosidase quantifications were carried out using ONPG assays as described in the CLONTECH Laboratories Yeast Protocols Handbook (protocol no. PT3024-1, version no. PR13103; Stratagene). The yeast strains were grown overnight at 30°C with shaking in 5 mL of –Trp/–Leu dropout media (0.7% yeast nitrogen base without amino acids, 0.1% mix of all essential amino acids excluding Trp and Leu). Two milliliters of overnight culture were then transferred to 8 mL of yeast peptone dextrose (YPD; 1% bacto-yeast extract, 2% bacto-peptone, 2% dextrose) media and grown at 30°C with shaking for approximately 2 h (OD600 of 1 mL = 0.5–1). Five replicates were separated from each culture. The yeast cells were pelleted by centrifugation and washed once with Z buffer (Na2HPO4, NaH2PO4, KCl, and MgSO4). The yeast cell pellets were then resuspended in Z buffer and were lysed using five sequential freeze thaw cycles involving transfers from liquid nitrogen to a 37°C water bath. A Z buffer/BME solution was added along with a Z buffer/ONPG solution. The samples were then incubated in a 30°C water bath and timed for rate of yellow coloration emergence. The ONPG reactions were quenched with Na2CO3, centrifuged at 20,800g for 10 min, and the OD420 was measured. β-Galactosidase activity was measured in Miller units (Miller, 1972): β-galactosidase units = 1,000 x OD420/(t x V x OD600); t = elapsed time (min) of ONPG reaction incubation; V = 0.1 mL x concentration factor; OD600 = A600 of 1 mL of culture after the approximately 2 h YPD grow up.
Yeast Two-Hybrid Growth Assays
Yeast cultures were grown as described above to yield OD600 of 1 mL equal to 0.5 to 1 in YPD media. A total of 1 x 108 cells of each culture were extracted and placed into a sterile tube. The cells were pelleted by centrifugation and were washed twice with –Leu/Trp/Ura/His (quadruple) dropout media. The final pellets were resuspended in 200 µL of quadruple dropout media. An aliquot of 5 µL of each culture was plated on quadruple dropout agar plates and growth was assessed after 6 d.
Coimmunoprecipitation
Coimmunoprecipitation was performed using a CO overexpression genotype. Briefly, the coding region of Arabidopsis CO was amplified using PCR from a cDNA template. The fragment was cloned into the pDONR221 entry vector using Gateway BP Clonase II and the Gateway LR Clonase II recombination reaction was used to directionally clone the insert into the overexpression vector pH7WG2D (Karimi et al., 2002). The pH7WG2D-CO vector was transferred into Agrobacterium strain GV3101 by electroporation. Plants were transformed by floral dipping as described for Res-mu. Transgenic seedlings were selected on 1x Murashige and Skoog media containing 35 mg/L hygromycin, and transgenicity was verified by the presence of the GFP visible marker in the pH7WG2D T-DNA region. Leaves of plants expressing 35S::CO were harvested at dusk by freezing in liquid nitrogen. Clarified extracts (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, and 1% Triton x100) were mixed with either Immunoprecipitin or Protein-A Sepharose, with or without the presence of anti-CO (no. sc-33753; Santa Cruz Biotechnology). Immunoprecipitin was collected and washed by centrifugation. Protein-A Sepharose was collected and washed by spin column. The washed collections were suspended in SDS-PAGE sample buffer for western blotting using the anti-14-3-3 antibody -GBC-4B9 (Sehnke et al., 2006).
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ACKNOWLEDGMENTS |
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and anti-µ 14-3-3 antibodies and Zhiyong Wang for immunoprecipitation insights. Received September 3, 2007; accepted September 29, 2007; published October 19, 2007.
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FOOTNOTES |
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The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert J. Ferl (robferl{at}ufl.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.108654
* Corresponding author; e-mail robferl{at}ufl.edu.
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Influence Transition to Flowering and Early Phytochrome Response
