Plant reproduction requires precise control of flowering in response to environmental cues. We isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vemalization or gibberellin treatment. We positionally cloned FKF1, which encodes a novel protein with a PAS domain similar to the flavin-binding region of certain photoreceptors, an F box characteristic of proteins that direct ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta propeller. FKF1 mRNA levels oscillate with a circadian rhythm, and deletion of FKF1 alters the waveform of rhythmic expression of two clock-controlled genes, implicating FKF1 in modulating the Arabidopsis circadian clock.

The phytohormone auxin is important in many aspects of plant development. We have isolated an auxin-resistant Arabidopsis mutant, iaa28-1 , that is severely defective in lateral root formation and that has diminished adult size and decreased apical dominance. The iaa28-1 mutant is resistant to inhibition of root elongation by auxin, cytokinin, and ethylene, but it responds normally to other phytohormones. We identified the gene defective in the iaa28-1 mutant by using a map-based positional approach and found it to encode a previously uncharacterized member of the Aux/IAA gene family. IAA28 is preferentially expressed in roots and inflorescence stems, and in contrast to other Aux/IAA genes, IAA28 transcription is not induced by exogenous auxin. Studies of the gain-of-function iaa28-1 mutant suggest that IAA28 normally represses transcription, perhaps of genes that promote lateral root initiation in response to auxin signals. INTRODUCTIONAuxins are plant growth hormones implicated in numerous developmental processes, including lateral root initiation, vascular tissue differentiation, establishment of apical dominance, and tropic responses (Davies, 1995). Many auxin responses result from changes in cell expansion and division, but these responses can vary in different tissues. For example, auxin promotes cell division in root pericycle cells, which leads to lateral root formation (Laskowski et al., 1995), but inhibits cell division in lateral meristems of the shoot, resulting in apical dominance (Hillman, 1984). The most common naturally occurring auxin is indoleacetic acid (IAA). Despite the importance of IAA in plant growth and development, the molecular details of auxin action remain largely unknown.Auxin rapidly and specifically alters transcript levels of numerous genes (Abel and Theologis, 1996), and many auxin effects may be mediated through changes in gene expression. Although numerous genes have been identified based on strong transcriptional responses to auxin, only a small number of these genes have well-understood functions. For instance, the Arabidopsis ACS4 gene, which encodes an ethylene biosynthetic enzyme, is rapidly induced by auxin (Abel et al., 1995a), which correlates with increased ethylene biosynthesis in response to auxin (Yang and Hoffman, 1984). The functions of the SAUR (for small auxin up RNA) genes, a family isolated on the basis of auxin-responsive transcription (McClure et al., 1989), are only beginning to be elucidated, but the observation that a maize SAUR protein binds calmodulin suggests a role for calcium in auxin signal transduction (Yang and Poovaiah, 2000).Aux/IAA family members were originally identified in pea because of their strong and rapid transcriptional induction in response to auxin (Theologis et al., 1985). On the basis of auxin-induced transcription and sequence homology, Aux/ IAA genes have also been isolated in several other species (Walker and Key, 1982;Ainley et al., 1988; Conner et al., 1990;Yamamoto et al., 1992;Abel et al., 1994). In Arabidopsis, nearly 20 ...

Attenuated activity of echinocandin antifungals at high concentrations, known as the "paradoxical effect," is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca 2؉ /calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the ⌬cnaA and ⌬crzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.The echinocandin antifungals inhibit 1,3-␤-D-glucan synthesis, and all three currently available echinocandins (caspofungin, anidulafungin, and micafungin) have activity against both yeasts and molds. However, in vitro and in vivo studies with this antifungal class have revealed an interesting "paradoxical effect" on growth, evidenced by a recurrent increase in the growth of the fungal organism at drug concentrations above a certain threshold (36).The paradoxical effect was first described for caspofungin treatment of Candida albicans and was not found to be due to mutation or increased expression of glucan synthase activity (30,32). Further studies showed that this paradoxical effect on growth occurred in many Candida species, appearing in both planktonic and biofilm cells (22). Early efforts to elucidate a mechanism revealed increased chitin content in the cell wall upon caspofungin treatment (31). Initial studies with the Saccharomyces cerevisiae FKS1 deletion mutant, a strain defective in the 1,3-␤-D-glucan synthase, showed that chitin biosynthesis is upregulated in response to glucan depletion (14). Furthermore, at the molecular level, several signal transduction pathways have been implicated in the regulation of the C. albicans paradoxical effect, including protein kinase C (PKC), highosmolarity glycerol response (HOG), and calcineurin signaling events (25,34,35). Alt...

Summary Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence, and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaA ΔcnaB strains of A. fumigatus were constructed. CnaA co-localizes to the contractile actin ring early during septation and remains at the center of the mature septum. While CnaB's septal localization is CnaA-dependent, CnaA's septal localization is CnaB-independent but CnaB is required for CnaA's function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaA ΔcnaB strain displayed more defective growth and aberrant septation. While three Ca2+-binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino-terminal arginine-rich domains (16-RRRR-19 and 44-RLRKR-48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51-KLDK-54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for calcineurin complex in regulating hyphal growth and septation.

The phytohormone auxin is important in many aspects of plant development. We have isolated an auxin-resistant Arabidopsis mutant, iaa28-1, that is severely defective in lateral root formation and that has diminished adult size and decreased apical dominance. The iaa28-1 mutant is resistant to inhibition of root elongation by auxin, cytokinin, and ethylene, but it responds normally to other phytohormones. We identified the gene defective in the iaa28-1 mutant by using a map-based positional approach and found it to encode a previously uncharacterized member of the Aux/IAA gene family. IAA28 is preferentially expressed in roots and inflorescence stems, and in contrast to other Aux/IAA genes, IAA28 transcription is not induced by exogenous auxin. Studies of the gain-of-function iaa28-1 mutant suggest that IAA28 normally represses transcription, perhaps of genes that promote lateral root initiation in response to auxin signals.