Differences in copper (Cu) absorption and transport, physiological responses and structural characteristics between two types of Cu-resistant plants, Oenothera glazioviana (Cu-exclusion type) and Elsholtzia haichowensis (Cu-enrichment type), were investigated in the present study. The results indicated the following: (1) After 50 μM Cu treatment, the Cu ratio in the xylem vessels of E. haichowensis increased by 60%. A Cu adsorption experiment indicated that O. glazioviana exhibited greater resistance to Cu, and Cu absorption and the shoot/root ratio of Cu were significantly lower in O. glazioviana than in E. haichowensis. (2) An analysis of the endogenous abscisic acid (ABA) variance and exogenous ABA treatment demonstrated that the ABA levels of both plants did not differ; exogenous ABA treatment clearly reduced Cu accumulation in both plants. (3) The leaf stomatal density of O. glazioviana was significantly less than that of E. haichowensis. Guard cells in E. haichowensis plants were covered with a thick cuticle layer, the epidermal hair was more numerous and longer, and the number of xylem conduits in the root was small. (4) The transpiration rate and the stomatal conductance of O. glazioviana were both significantly lower than those of E. haichowensis, regardless of whether the plants were treated with Cu. Taken together, these results indicate that the differences in the structural characteristics between these two plant species, particularly in the characteristics related to plant transpiration, are important factors that govern whether plants acquire or exclude Cu.

In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone h2a.z, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm.

Circadian rhythms are pervasive among eukaryotes, and the underlying clocks share a common regulatory architecture—a negative feedback loop. A wealth of genetic and biochemical data underpin current perceptions of circadian oscillators but aspects of their cell biology remain cryptic, especially in syncytial systems like Neurospora crassa. We employed novel microfluidic systems and a light-blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles in Neurospora. Despite heterogeneity of clock gene (frq) expression, we find robust, synchronous cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form small, highly dynamic nuclear bodies that persist throughout the cycle and exhibit time-dependent changes in composition, including transient colocalization between the positive and negative components for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how dynamic subnuclear organization and internuclear exchange of clock proteins ensure synchronous regulation of cellular activities across a macroscopic, multinucleated syncytium.

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is mainly controlled by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.