Woe-Yeon Kim

Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana

The underlying mechanism of circadian rhythmicity appears to be conserved among organisms, and is based on negative transcriptional feedback loops forming a cellular oscillator (or ‘clock’). Circadian changes in protein stability, phosphorylation and subcellular localization also contribute to the generation and maintenance of this clock. In plants, several genes have been shown to be closely associated with the circadian system. However, the molecular mechanisms proposed to regulate the plant clock are mostly based on regulation at the transcriptional level. Here we provide genetic and molecular evidence for a role of ZEITLUPE (ZTL) in the targeted degradation of TIMING OF CAB EXPRESSION 1 (TOC1) in Arabidopsis thaliana (thale cress). The physical interaction of TOC1 with ZTL is abolished by the ztl-1 mutation, resulting in constitutive levels of TOC1 protein expression. The dark-dependent degradation of TOC1 protein requires functional ZTL, and is prevented by inhibiting the proteosome pathway. Our results show that the TOC1–ZTL interaction is important in the control of TOC1 protein stability, and is probably responsible for the regulation of circadian period by the clock.

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

As an F-box protein, ZEITLUPE (ZTL) is involved in targeting one or more substrates for ubiquitination and degradation via the proteasome. The initial characterization of ZTL suggested a function limited largely to the regulation of the circadian clock. Here, we show a considerably broader role for ZTL in the control of circadian period and photomorphogenesis. Using a ZTL-specific antibody, we quantitated and characterized a ZTL dosage series that ranges from a null mutation to a strong ZTL overexpressor. In the dark, ztl null mutations lengthen circadian period, and overexpression causes arrhythmicity, suggesting a more comprehensive role for this protein in the clock than previously suspected. In the light, circadian period becomes increasingly shorter at higher levels of ZTL, to the point of arrhythmicity. By contrast, hypocotyl length increases and flowering time is delayed in direct proportion to the level of ZTL. We propose a novel testable mechanism by which circadian period and amplitude may act together to gate phytochrome B–mediated suppression of hypocotyl. We also demonstrate that ZTL-dependent delay of flowering is mediated through decreases in CONSTANS and FLOWERING LOCUS T message levels, thus directly linking proteasome-dependent proteolysis to flowering.

The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure

Loss-of-function siz1 mutations caused early flowering under short days. siz1 plants have elevated salicylic acid (SA) levels, which are restored to wild-type levels by expressing nahG, bacterial salicylate hydroxylase. The early flowering of siz1 was suppressed by expressing nahG, indicating that SIZ1 represses the transition to flowering mainly through suppressing SA-dependent floral promotion signaling under short days. Previous results have shown that exogenous SA treatment does not suppress late flowering of autonomous pathway mutants. However, the siz1 mutation accelerated flowering time of an autonomous pathway mutant, luminidependens, by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor. This result suggests that SIZ1 promotes FLC expression, possibly through an SA-independent pathway. Evidence indicates that SIZ1 is required for the full activation of FLC expression in the late-flowering FRIGIDA background. Interestingly, increased FLC expression and late flowering of an autonomous pathway mutant, flowering locus d (fld), was not suppressed by siz1, suggesting that SIZ1 promotes FLC expression by repressing FLD. Consistent with this, SIZ1 facilitates sumoylation of FLD that can be suppressed by mutations in three predicted sumoylation motifs in FLD (i.e. FLDK3R). Furthermore, expression of FLDK3R in fld protoplasts strongly reduced FLC transcription compared with expression of FLD, and this affect was linked to reduced acetylation of histone 4 in FLC chromatin. Taken together, the results suggest that SIZ1 is a floral repressor that not only represses the SA-dependent pathway, but also promotes FLC expression by repressing FLD activity through sumoylation, which is required for full FLC expression in a FRIGIDA background.

Activation of the plasma membrane Na/H antiporter Salt-Overly-Sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2–SOS3 calcium-dependent protein kinase complex up-regulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2–SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2–SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

Circadian phase-specific degradation of the F-box protein ZTL is mediated by the proteasome

Critical to the maintenance of circadian rhythmicity is the cyclic expression of at least some components of the central oscillator. High-amplitude cycling of mRNA and protein abundance, protein phosphorylation and nuclear/cytoplasmic shuttling have all been implicated in the maintenance of circadian period. Here we use a newly characterized Arabidopsis suspension cell culture to establish that the rhythmic changes in the levels of the clock-associated F-box protein, ZTL, are posttranscriptionally controlled through different circadian phase-specific degradation rates. This proteolysis is proteasome dependent, implicating ZTL itself as substrate for ubiquitination. This demonstration of circadian phase-regulated degradation of an F-box protein, which itself controls circadian period, suggests a novel regulatory feedback mechanism among known circadian systems.

Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis

Environmental challenges to plants typically entail retardation of vegetative growth and delay or cessation of flowering. Here we report a link between the flowering time regulator, GIGANTEA (GI), and adaptation to salt stress that is mechanistically based on GI degradation under saline conditions, thus retarding flowering. GI, a switch in photoperiodicity and circadian clock control, and the SNF1-related protein kinase SOS2 functionally interact. In the absence of stress, the GI:SOS2 complex prevents SOS2-based activation of SOS1, the major plant Na(+)/H(+)-antiporter mediating adaptation to salinity. GI overexpressing, rapidly flowering, plants show enhanced salt sensitivity, whereas gi mutants exhibit enhanced salt tolerance and delayed flowering. Salt-induced degradation of GI confers salt tolerance by the release of the SOS2 kinase. The GI-SOS2 interaction introduces a higher order regulatory circuit that can explain in molecular terms, the long observed connection between floral transition and adaptive environmental stress tolerance in Arabidopsis.

Forward Genetic Analysis of the Circadian Clock Separates the Multiple Functions of ZEITLUPE

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.

Independent Roles for EARLY FLOWERING 3 and ZEITLUPE in the Control of Circadian Timing, Hypocotyl Length, and Flowering Time

The circadian clock regulates many aspects of plant development, including hypocotyl elongation and photoperiodic induction of flowering. ZEITLUPE (ZTL) is a clock-related F-box protein, and altered ZTL expression causes fluence rate-dependent circadian period effects, and altered hypocotyl elongation and flowering time. EARLY FLOWERING 3 (ELF3) is a novel protein of unknown biochemical function. elf3 mutations cause light-dependent circadian dysfunction, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is unclear. Here we show that the effects of ZTL and ELF3 on circadian clock function and early photomorphogenesis are additive. The long period of ztl mutations and ELF3 overexpressors are more severe than either alone. Dark-release experiments showing additivity in phase advances suggest that the arrthymicity caused by ZTL overexpression and that of the elf3-1 mutation arise through independent pathways. A similar additive effect on hypocotyl elongation in red and blue light is also observed. In contrast, ELF3 and ZTL overexpressors act similarly to control flowering time in long days through the CONSTANS/FLOWERING LOCUS T (CO/FT) pathway. ZTL overexpression does not delay flowering through changes in GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F-BOX levels, but through a ZTL-mediated reduction in CO expression. In contrast, ELF3 negatively regulates CO, FT, and GIGANTEA transcript levels, as the expression of all three genes is increased in elf3-1. The elf3-1 co-1 double mutant flowers much earlier in long days than co-1, although FT message levels remain very low. These results show that elf3-1 can derepress late flowering through a CO-independent mechanism. ELF3 may act at more than one juncture, possibly posttranscriptionally.

TsHKT1;2, a HKT1 Homolog from the Extremophile Arabidopsis Relative Thellungiella salsuginea, Shows K+ Specificity in the Presence of NaCl

Cellular Na+/K+ ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na+/K+ cotransporters in the High-affinity K+ Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na+ sensitivity and K+ deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na+ and showed less K+ deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na+ or K+ transporters revealed strong K+ transporter activity and selectivity for K+ over Na+. Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K+ transporter activity. We consider the maintenance of K+ uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

Overexpression of Arabidopsis YUCCA6 in Potato Results in High-Auxin Developmental Phenotypes and Enhanced Resistance to Water Deficit

Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.