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F-Box Proteins Take Center Stage

neckardt{at}aspb.org

It has long been recognized that protein turnover is crucial for the maintenance of cellular homeostasis. Since the characterization of the ubiquitin (Ub)/26S proteasome pathway and its recognition as a major route of protein degradation in eukaryotes (Hershko and Ciechanover, 1998; Deshaies, 1999), it has become increasingly apparent that the long arm of death reaches into nearly every aspect of life. Arabidopsis (Arabidopsis thaliana) encodes at least 1300 genes that encode core components of the Ub/26S proteasome pathway, illustrating that death (degradation) and birth processes (transcription and translation) are equal and opposite sides of the coin in the life of proteins.

Two major protein complexes participate in the Ub/26S proteasome system, the SKP1-Cullin-F-box protein (SCF) E3 ligase complex, which marks target proteins for degradation via the ATP-dependent covalent attachment of Ub, and the 26S proteasome complex, which recognizes and degrades ubiquitinated substrates. The F-box protein subunit carries the specificity for recognition and binding of target proteins and anchors them in the proper orientation within the SCF complex for subsequent ubiquitination via activity of other SCF core subunits. The Arabidopsis genome encodes nearly 700 different F-box proteins (and also several distinct SKP1- and Cullin-type subunits), suggesting that plants have the capability of generating an enormous collection of distinct SCF subtypes with unique specificities and unique functions in plant growth and development (Vierstra, 2003). In this issue of The Plant Cell, two independent reports provide evidence that F-box proteins play key roles in the regulation of self-incompatibility (Qiao et al., pages 582–595) and in control of the circadian clock, photomorphogenesis, and flowering time (Somers et al., pages 769–782).

F-Box Proteins and Self-Incompatibility
Self-incompatibility (SI) allows the pistil of a flower to recognize and reject self-pollen. It is an effective mechanism for preventing inbreeding and promoting outcrossing, especially for hermaphroditic plants, and is widespread among flowering plants. The S-RNase–based mechanism of SI, or Solanaceae type, is the most widely studied. In addition to being found in the Solanaceae (Solanum, Lycopersicon, Nicotiana, and Petunia), this type is also found in the Rosaceae (Malus, Prunus, and Pyrus) and Scrophulariaceae (Antirrhinum) (Kao and Tsukamoto, 2004). Most cultivated species in these families are self-compatible because SI was selected out in breeding programs designed to produce inbred lines homozygous for desirable crop traits, and the mechanism is studied mainly in noncultivated species.

The SI response is controlled by the S-locus, which is highly polymorphic and contains distinct genes for male and female specificity (reviewed in McCubbin and Kao, 2000; Wang et al., 2003; Kao and Tsukamoto, 2004). Haploid pollen carrying an S-haplotype identical to one of two S-haplotypes carried by the diploid pistil is recognized as self-pollen and is rejected. The S-RNase gene controls female specificity at the S-locus, and RNase activity is essential for the SI response (Huang et al., 1994). It is generally accepted that S-RNases act to degrade RNA of self-pollen tubes resulting in the inhibition of fertilization, but many details of the mechanism are unknown.

One of the major unknown details is the identity of the male specificity or pollen S-gene. Clusters of F-box genes termed S-locus F-box (SLF) genes have been found very tightly linked to the S-RNase gene in many plants, including Prunus species and Antirrhinum, and are viewed as candidates for the pollen S-gene (reviewed in Kao and Tsukamoto, 2004). For example, PmSLF in Prunus mume lies within the S-locus close to the S-RNase gene, shows a high level of polymorphism, and is expressed in pollen (Entani et al., 2003). Antirrhinum AhSLF shows some degree of polymorphism (although considerably less than the Rosaceae SLF genes) and is expressed specifically in pollen and tapetum cells (Lai et al., 2002; Zhou et al., 2003). F-box proteins are predicted to target substrate proteins for degradation via the Ub/26S proteasome pathway, but the function of SLF proteins has not been demonstrated.

Two models have been proposed to explain how S-RNases and pollen S-allele gene products function to control the specificity of SI (reviewed in Kao and Tsukamoto, 2004). The inhibitor model predicts that the pollen S-gene encodes an RNase inhibitor (or protein associated with RNase inhibition) whose function is blocked during the SI response, whereas the receptor model predicts that the pollen S-gene encodes a membrane or cell wall receptor that only allows S-RNases of the matching haplotype to enter a pollen tube. The available data support an inhibitor model because S-RNases have been identified in the cytoplasm of self-pollen as well as non-self pollen tubes (Luu et al., 2000).

The data presented by Qiao et al. provide additional evidence that both S-RNases and SLF proteins are fundamental to the SI response. The authors used coimmunoprecipitation and yeast two-hybrid assays to show that AhSLF-S₂ interacts with S-RNases and also interacts with ASK1- and CULLIN1-like proteins that together likely form an SCF complex that targets S-RNases for degradation. They further showed that compatible (non-self) pollination was blocked after treatment with proteasomal inhibitors, whereas the incompatible pollination reaction was unaffected, supporting the notion that 26S proteasome activity is required for compatible pollination. Finally, a series of experiments showed that the ubiquitination level of style proteins was higher, and S-RNases appear to be subsequently reduced after compatible pollination relative to incompatible pollination reactions.

But does SLF constitute the pollen S-gene? Under the inhibitor model, it is difficult to envision how SLF could function as the pollen S-gene because a direct interaction between the pollen S-gene product and S-RNases is required for the self-incompatible reaction (McCubbin and Kao, 2000), whereas these data clearly implicate a direct interaction between SLF protein and S-RNases in the compatible (non-self) pollination reaction. In other words, how does SLF (or any other pollen S-gene product) inhibit all S-RNases except for the self S-RNase? Another candidate for pollen S might be a protein that interacts with S-RNase to prevent the interaction with SLF or the activity of the SCF complex. For example, most of the known targets of F-box proteins require phosphorylation for F-box recognition (Vierstra, 2003). Under this scenario, pollen S could be a phosphatase or other protein affecting the phosphorylation status of S-RNases. However, it is recognized that the pollen S-gene must be genetically tightly linked to the S-RNase gene, and it must show a significant degree of polymorphism to determine S-haplotype specificity (McCubbin and Kao, 2000). The SLF genes that have been identified share these features, suggesting that they participate in determining specificity. Furthermore, if pollen S were a separate gene, this would imply the existence of three tightly linked genes encoding S-haplotype specificity (S-RNase, SLF, and pollen S), and it is difficult to explain the evolutionary maintenance of such a mechanism.

One scenario that might explain how SLF genes could encode pollen S-haplotype specificities would be that SLF proteins interact with S-RNases in both compatible and self-incompatible pollination reactions, but the interaction between proteins sharing identical S-haplotypes (self-incompatible) would be unable to form a functional SCF complex, and the S-RNase would remain active. This scenario is consistent with the simple inhibitor model (Kao and Tsukamoto, 2004), which proposes that pollen S products contain both an S-allele specificity domain and an inhibitor domain. In this model, the inhibitor domain interacts nonspecifically with a common domain of all non-self RNases, but this interaction is prevented by the interaction of the specificity domain with the matching S-allele specificity domain of self RNases. The results of Qiao et al. support this idea because in vitro and in vivo protein interaction experiments showed that AhSLF-S₂ interacts nonspecifically with S-RNases of various S-haplotype specificities. A key question is whether SLF protein interacts with self and non-self RNase differently, rendering only the former immune to Ub/26S proteasome–mediated degradation. The work of Qiao et al. supports this notion because additional experiments showed that Ub/26S proteasome activity, ubiquitination of S-RNases, and degradation of S-RNases are associated with compatible and not self-incompatible pollination reactions. Thus, the work of Qiao et al. provides important new information on S-RNase–type SI that should help guide efforts to solve the identity of the pollen S-gene and determine the precise functions of SLF proteins and S-RNases in this system.

F-Box Proteins and the Circadian Clock
The circadian clock influences a wide range of developmental processes in plants, including photomorphogenesis and flowering time. The clock consists of a central oscillator connected to signal transduction input pathways that entrain the clock to prevailing environmental conditions (e.g., daylength and temperature) and to output pathways that regulate responses controlled by the clock (Barak et al., 2000; McClung, 2001). The higher plant circadian clock is most well characterized in Arabidopsis. A principal component of the central oscillator is believed to be a negative feedback loop that includes the pseudoresponse regulator protein TIMING OF CAB1 (TOC1) and the myb transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1). Phytochrome and cryptochrome photoreceptors provide major inputs to the clock, and a major output pathway controlling flowering time is regulated by CONSTANS (CO) and FLOWERING LOCUS T (FT) (reviewed in Hamaya and Coupland, 2003).

The F-box protein ZEITLUPE (ZTL) has been identified as an additional component of light-input pathways to the circadian clock. The ztl mutant exhibits an increased period length in the circadian rhythm of CHLOROPHYLL A/B BINDING PROTEIN (CAB) gene expression (Somers et al., 2000), and ZTL has been shown to interact with the photoreceptors PhyB and Cry1 in vitro (Jarillo et al., 2001). Interestingly, ZTL has a photoreceptor-like LOV domain, similar to the photoreceptor domain of phototropins, further supporting the idea that it regulates some aspect of light input to the circadian clock. In the present study, Somers et al. show that ZTL affects the phasing of CCA1 and TOC1 gene expression, which is essential for normal clock function and the regulation of output pathways. The authors also provide evidence of a relationship between ZTL and PhyB in control of hypocotyl expansion and show that ZTL can act to repress CO and FT gene expression and to delay flowering.

The authors examined circadian periods in Arabidopsis in ztl null mutant plants and in a series of transgenic plant lines exhibiting different levels of ZTL overexpression. Circadian periods were monitored using clock-regulated CAB:luciferase (luc) and COLD CIRCADIAN RHYTHM-RNA BINDING2:luc reporter genes. First, it was shown that ztl null mutations increased the length of the free-running circadian period, and overexpression of ZTL shortened the period by several hours relative to the wild type. The strongest ZTL overexpression line exhibited either complete arrhythmicity or a very short period with low amplitude cycling. Overall, the results showed that the circadian period is extremely sensitive to ZTL levels (i.e., less than twofold change), and there is a dosage-dependent effect of ZTL on period.

The authors then examined the effect of ZTL on two developmental processes influenced by the circadian clock: hypocotyl expansion and flowering time. Somers et al. (2000) previously showed that the ztl mutation causes hypersensitivity of hypocotyl expansion to red light. In the current work, the authors show that overexpression of ZTL resulted in hypocotyl lengthening, particularly under red light. Although phyB provides input to the pathway controlling hypocotyl expansion and ZTL previously has been shown to interact with phyB (Jarillo et al., 2001), the data of Somers et al. suggests that ZTL did not interfere with phyB signaling. The authors propose that ZTL activity might influence phyB-mediated clock-controlled hypocotyl expansion in a gated fashion rather than as a simple on–off switch. The interaction of ZTL and phyB is being further investigated with the use of phyB mutant plants transformed to overexpress ZTL.

The overexpression of ZTL also caused a significant delay in flowering time under long days. Transcript levels of two principal clock-controlled flowering time regulatory genes, CO and FT, were strongly suppressed in the plants overexpressing ZTL, suggesting that ZTL might suppress flowering time by inhibiting the expression of these genes. Interestingly, a ZTL-overexpressing line exhibiting an arrhythmic period (and very strong overexpression of ZTL) and a line that cycled with a very short period (with modest ZTL overexpression) both showed the same degree of delayed flowering in long days, raising the possibility that the effect of ZTL on flowering time is independent of the clock.

One of the major questions remaining is what proteins are targeted by ZTL for degradation via the Ub/26S proteasome pathway to produce these effects? A series of experiments that examined CCA1 and TOC1 mRNA transcript abundance and, in some conditions, protein levels showed that ZTL affects the normal phasing of expression of these two principal components of the central oscillator. The authors argue that ZTL for the most part does not affect transcription or transcript stability of CCA1 and TOC1, suggesting that it does not target transcription factors that directly regulate these genes. The results also suggest that CCA1 itself is not a target of ZTL because ztl loss-of-function mutations result in a significant reduction in CCA1 protein levels. Mas et al. (2003) recently showed that TOC1 protein interacts with ZTL, and ztl mutant plants show increased levels of TOC1 protein relative to the wild type, suggesting that TOC1 is likely targeted for Ub/26S proteasome–mediated degradation by ZTL.

CCA1 and LHY are believed to operate in a negative feedback loop with TOC1, whereby TOC1 functions as a positive regulator of CCA1 and LHY expression and CCA1 and LHY proteins in turn suppress TOC1 expression. Therefore, ztl mutant plants might be expected to show increased CCA1 protein levels because of increased TOC1. The observation that they exhibit a significant decrease in CCA1 protein and mRNA levels suggests that the regulation of CCA1 involves more than simple positive upregulation by TOC1.

Coordinated phasing of components of the central oscillator is essential to regulate the oscillatory behavior of the clock, which is critical to clock function. Perturbations to this system likely induce numerous indirect effects, making it difficult to determine the functions of all of the interacting partners. The work of Somers et al. confirms that ZTL is a key player in regulation of the circadian clock, in turn affecting clock outputs such as hypocotyl expansion and the control of flowering time. Imaizumi et al. (2003) have shown that FLAVIN BINDING KELCH REPEAT F-BOX1 protein, which shares sequence similarity with ZTL, is influenced by the circadian clock and participates in the regulation of CO expression and flowering time. These results, together with those of Somers et al., establish the importance of the Ub/26S proteasome pathway in the control of the circadian clock and regulation of flowering time.

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Related articles in Plant Cell:

The F-Box Protein AhSLF-S₂ Physically Interacts with S-RNases That May Be Inhibited by the Ubiquitin/26S Proteasome Pathway of Protein Degradation during Compatible Pollination in Antirrhinum

Hong Qiao, Hongyun Wang, Lan Zhao, Junli Zhou, Jian Huang, Yansheng Zhang, and Yongbiao Xue
Plant Cell 2004 16: 582-595. [Abstract] [Full Text]  

The F-Box Protein ZEITLUPE Confers Dosage-Dependent Control on the Circadian Clock, Photomorphogenesis, and Flowering Time

David E. Somers, Woe-Yeon Kim, and Ruishuang Geng
Plant Cell 2004 16: 769-782. [Abstract] [Full Text]  

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