Takatoshi Kiba

PSEUDO-RESPONSE REGULATORS 9, 7, and 5 Are Transcriptional Repressors in the Arabidopsis Circadian Clock

PSEUDORESPONSE REGULATOR9 (PRR9), PRR7, and PRR5 regulate the Arabidopsis circadian clock. PRR9, PRR7, and PRR5 proteins associate with CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL promoters in a sequential manner to repress their expression. A conserved region common to the three PRR proteins is sufficient for repressor activity. An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

Expression of Arabidopsis response regulator homologs is induced by cytokinins and nitrate

We examined cytokinin and nitrate responsiveness in gene expression of five distinct response regulator homologs (ARR3–ARR7) in the leaves of nitrogen‐starved Arabidopsis plants. The transcripts accumulated after spraying the shoots with t‐zeatin. The induction of accumulation was highly specific for cytokinins. The transcripts also accumulated by supply of nitrate to the culture medium. These findings suggest that ARRs are involved in inorganic nitrogen signal transduction mediated by cytokinin as in the case of ZmCip1, a response regulator homolog recently identified in maize.

Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin

Nitrogen is the mineral nutrient that often limits plant growth and development. In response to changes in nitrogen supply, plants display elaborate responses at both physiological and morphological levels to adjust their growth and development. Because higher plants consist of multiple organs with different functions and nutritional requirements, they rely on local and long-distance signalling pathways to coordinate the responses at the whole-plant level. Phytohormones have been considered as signalling substances of such pathways. Amongst phytohormones, abscisic acid, auxin, and cytokinins have been closely linked to nitrogen signalling. Recent evidence has provided some insights into how nitrogen and the phytohormone signals are integrated to bring about changes in physiology and morphology. In this review, the evidence is summarized, mostly focusing on examples related to nitrogen acquisition.

Targeted Degradation of PSEUDO-RESPONSE REGULATOR5 by an SCFZTL Complex Regulates Clock Function and Photomorphogenesis in Arabidopsis thaliana

Circadian clocks comprise several regulatory feedback loops that control gene transcription. However, recent evidence has shown that posttranslational mechanisms are also required for clock function. In Arabidopsis thaliana, members of the PSEUDO-RESPONSE REGULATOR (PRR) family were proposed to be components of the central oscillator. Using a PRR5-specific antibody, we characterized changes in PRR5 protein levels in relation to its mRNA levels under various circadian conditions. Under long-day conditions, PRR5 mRNA levels are undetectable at dusk but PRR5 protein levels remain maximal. Upon dark transition, however, PRR5 levels decrease rapidly, indicating dark-induced, posttranslational regulation. We demonstrated that the Pseudo-Receiver (PR) domain of PRR5 interacts directly with the F box protein ZEITLUPE (ZTL) in vitro and in vivo. Analyses of mutants and transgenic plants revealed an inverse correlation between PRR5 and ZTL levels, which depends on the PR domain. These results indicate that PRR5 is negatively regulated by ZTL, which likely mediates its ubiquitination and degradation. Phenotypic analyses of prr5 ztl double mutants showed that PRR5 is required for ZTL functions. ZTL contains a Light-Oxygen-Voltage domain, and its activity may be directly regulated by blue light. Consistent with this notion, we found that blue light stabilizes PRR5, although it does not alter ZTL levels. Together, our results show that ZTL targets PRR5 for degradation by 26S proteasomes in the circadian clock and in early photomorphogenesis.

Metabolism and Long‐distance Translocation of Cytokinins

During plant development, distantly-located organs must communicate in order to adapt morphological and physiological features in response to environmental inputs. Among the recognized signaling molecules, a class of phytohormones known as the cytokinins functions as both local and long-distance regulatory signals for the coordination of plant development. This cytokinin-dependent communication system consists of orchestrated regulation of the metabolism, translocation, and signal transduction of this phytohormone class. Here, to gain insight into this elaborate signaling system, we summarize current models of biosynthesis, trans-membrane transport, and long-distance translocation of cytokinins in higher plants.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

In Arabidopsis thaliana, the F-box protein ZTL affects the period length of the circadian clock by regulating the stability of the core clock proteins TOC1 and PRR5. This study shows that, together with ZTL, the ZTL homologs FKF1 and LKP2 are also involved in the same protein stability regulation to determine the pace and robustness of the plant circadian clock. Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

The Arabidopsis Nitrate Transporter NRT2.4 Plays a Double Role in Roots and Shoots of Nitrogen-Starved Plants

The NITRATE TRANSPORTER2.4 (NRT2.4) is a member of the NRT2 gene family in Arabidopsis thaliana and is preferentially expressed under nitrogen starvation. This study shows that NRT2.4 is a nitrate transporter involved both in root nitrate uptake at very low nitrate concentration and in delivering nitrate to the shoot phloem under nitrogen starvation. Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.

Transcriptional repressor PRR5 directly regulates clock-output pathways

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.

Arabidopsis ABCG14 is essential for the root-to-shoot translocation of cytokinin

Significance Roots and shoots communicate with each other to synchronize and optimize plant growth and respond to environmental changes. Shoots and roots exchange signals to sense the status and respond to the needs of the other organ. Cytokinins, which are phytohormones that regulate various aspects of growth and development, are recognized as the most important signal transmitted from roots to shoots. Whereas the enzymes underlying cytokinin biosynthesis and the corresponding receptors have been identified, our knowledge of cytokinin transport is limited. In this study, we identified the Arabidopsis ATP-binding cassette transporter subfamily G14 as a major component in the transfer of cytokinins from roots to shoots and hence as a regulator of shoot development. This finding represents a major breakthrough in the field. Cytokinins are phytohormones that induce cytokinesis and are essential for diverse developmental and physiological processes in plants. Cytokinins of the trans-zeatin type are mainly synthesized in root vasculature and transported to the shoot, where they regulate shoot growth. However, the mechanism of long-distance transport of cytokinin was hitherto unknown. Here, we report that the Arabidopsis ATP-binding cassette (ABC) transporter subfamily G14 (AtABCG14) is mainly expressed in roots and plays a major role in delivering cytokinins to the shoot. Loss of AtABCG14 expression resulted in severe shoot growth retardation, which was rescued by exogenous trans-zeatin application. Cytokinin content was decreased in the shoots of atabcg14 plants and increased in the roots, with consistent changes in the expression of cytokinin-responsive genes. Grafting of atabcg14 scions onto wild-type rootstocks restored shoot growth, whereas wild-type scions grafted onto atabcg14 rootstocks exhibited shoot growth retardation similar to that of atabcg14. Cytokinin concentrations in the xylem are reduced by ∼90% in the atabcg14 mutant. These results indicate that AtABCG14 is crucial for the translocation of cytokinin to the shoot. Our results provide molecular evidence for the long-distance transport of cytokinin and show that this transport is necessary for normal shoot development.

Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions

As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.