PSEUDO-RESPONSE REGULATOR Research Articles

Cold-induced degradation of core clock proteins implements temperature compensation in the Arabidopsis circadian clock.

The period of circadian clocks is maintained at close to 24 hours over a broad range of physiological temperatures due to temperature compensation of period length. Here, we show that the quantitative control of the core clock proteins TIMING OF CAB EXPRESSION 1 [TOC1; also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)] and PRR5 is crucial for temperature compensation in Arabidopsis thaliana. The prr5 toc1 double mutant has a shortened period at higher temperatures, resulting in weak temperature compensation. Low ambient temperature reduces amounts of PRR5 and TOC1. In low-temperature conditions, PRR5 and TOC1 interact with LOV KELCH PROTEIN 2 (LKP2), a component of the E3 ubiquitin ligase Skp, Cullin, F-box (SCF) complex. The lkp2 mutations attenuate low temperature-induced decrease of PRR5 and TOC1, and the mutants display longer period only at lower temperatures. Our findings reveal that the circadian clock maintains its period length despite ambient temperature fluctuations through temperature- and LKP2-dependent control of PRR5 and TOC1 abundance.

Genome-wide identification of clock-associated genes and circadian rhythms in Fragaria × ananassa seedlings

Flowering time in plants is regulated by a photoperiod-responsive mechanism. Some plant species use a circadian clock-based control mechanism to adapt to variable environments. Strawberry is a horticultural crop that responds to certain photoperiods and temperatures to induce flowering. However, clock-associated genes in octoploid cultivated strawberry (Fragaria × ananassa) have not been defined, and their regulatory mechanism for responding to photoperiods is unclear. We herein targeted 12 clock-associated genes reported in other plant species and performed a genome-wide analysis and expression comparison in F. × ananassa seedlings. Seventy-eight sequences were selected from the F. × ananassa genome. The major domains and cis-acting elements were conserved in each sequence. Transcripts were clearly expressed under continuous light conditions in F. × ananassa seedlings (‘Yotsuboshi’) acclimated to long days. Among them, 9 genes maintained their unique autonomous circadian rhythms and may function as clock genes. LHY (LATE ELONGATED HYPOCOTYL) had the Myb domain and LHY expression peaked in the dawn. PRR (PSEUDO-RESPONSE REGULATOR) family members (PRR9, PRR7, PRR5, and TOC1 (TIMING OF CAB EXPRESSION 1)) had a pseudo-receiver domain and CCT domain, and peak expression times began sequentially from the afternoon for PRR9 to the evening for TOC1. LUX (LUXARRHYTHMO) had a Myb domain, and LUX expression peaked in evening with ELF3 (EARLY FLOWERING 3). FKF1 (FLAVIN-BINDING KELCH REPEAT F BOX 1) had PAS and F-box domains, and FKF1 expression peaked in the afternoon. GI (GIGANTEA) expression also peaked in the afternoon. F. × ananassa (‘Yotsuboshi’) appears to have multiple feedback loops comprising clock-associated genes. Although the rhythmic expression of CHE (CCA1 HIKING EXPEDITION) and ZTL (ZEITLUPE) was not observed, they had conserved domains, CHE with the TCP domain and ZTL with the PAS and F-box domains. The present results provide basic information on the circadian clock for the control of F. × ananassa flowering.

Nuclear interactions between the Pseudo-Response Regulator clock proteins and the Multi-Step Phosphorelay mediator Histidine-containing phosphotransfers in the moss Physcomitrium patens

Pseudo-Response Regulator (PRR) proteins constitute a fundamental set of circadian clock components in plants. PRRs have an amino acid sequence stretch with similarity to the receiver (REC) domain of response regulators (RRs) in the Multi-Step Phosphorelay (MSP). However, it has never been elucidated whether PRRs interact with Histidine-containing Phosphotransfer (HPt) proteins, which transfer a phosphate to RRs. Here, we studied whether PRRs interact with HPts in the moss Physcomitrium patens by the Yeast Two-Hybrid system and Bimolecular Fluorescence Complementation. P. patens PRR1/2/3 interacted with HPt1/2 in the nucleus, but not with HPt3, suggesting that P. patens PRRs function as authentic RRs. We discuss these results in relation to the evolution and diversity of the plant circadian clocks.

Molecular characterization of PSEUDO RESPONSE REGULATOR family in Rosaceae and function of PbPRR59a and PbPRR59b in flowering regulation

BackgroundPSEUDO RESPONSE REGULATOR (PRR) genes are essential components of circadian clock, playing vital roles in multiple processes including plant growth, flowering and stress response. Nonetheless, little is known about the evolution and function of PRR family in Rosaceae species.ResultsIn this study, a total of 43 PRR genes in seven Rosaceae species were identified through comprehensive analysis. The evolutionary relationships were analyzed with phylogenetic tree, duplication events and synteny. PRR genes were classified into three groups (PRR1, PRR5/9, PRR3/7). The expansion of PRR family was mainly derived from dispersed and whole-genome duplication events. Purifying selection was the major force for PRR family evolution. Synteny analysis indicated the existence of multiple orthologous PRR gene pairs between pear and other Rosaceae species. Moreover, the conserved motifs of eight PbPRR proteins supported the phylogenetic relationship. PRR genes showed diverse expression pattern in various tissues of pear (Pyrus bretschneideri). Transcript analysis under 12-h light/ dark cycle and constant light conditions revealed that PRR genes exhibited distinct rhythmic oscillations in pear. PbPRR59a and PbPRR59b highly homologous to AtPRR5 and AtPRR9 were cloned for further functional verification. PbPRR59a and PbPRR59b proteins were localized in the nucleus. The ectopic overexpression of PbPRR59a and PbPRR59b significantly delayed flowering in Arabidopsis transgenic plants by repress the expression of AtGI, AtCO and AtFT under long-day conditions.ConclusionsThese results provide information for exploring the evolution of PRR genes in plants, and contribute to the subsequent functional studies of PRR genes in pear and other Rosaceae species.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown. We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR1 (CBF1) and COLD-REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance.

AGAMOUS-LIKE 24 senses continuous inductive photoperiod in the inflorescence meristem to promote anthesis in chrysanthemum.

During the floral transition, many plant species including chrysanthemum (Chrysanthemum morifolium) require continuous photoperiodic stimulation for successful anthesis. Insufficient photoperiodic stimulation results in flower bud arrest or even failure. The molecular mechanisms underlying how continuous photoperiodic stimulation promotes anthesis are not well understood. Here, we reveal that in wild chrysanthemum (Chrysanthemum indicum), an obligate short-day (SD) plant, floral evocation is not limited to SD conditions. However, SD signals generated locally in the inflorescence meristem (IM) play a vital role in ensuring anthesis after floral commitment. Genetic analyses indicate that the florigen FLOWERING LOCUS T-LIKE3 (CiFTL3) plays an important role in floral evocation, but a lesser role in anthesis. Importantly, our data demonstrate that AGAMOUS-LIKE 24 (CiAGL24) is a critical component of SD signal perception in the IM to promote successful anthesis, and that floral evocation and anthesis are two separate developmental events in chrysanthemum. We further reveal that the central circadian clock component PSEUDO-RESPONSE REGULATOR 7 (CiPRR7) in the IM activates CiAGL24 expression in response to SD conditions. Moreover, our findings elucidate a negative feedback loop in which CiAGL24 and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (CiSOC1) modulate LEAFY (CiLFY) expression. Together, our results demonstrate that the CiPRR7-CiAGL24 module is vital for sustained SD signal perception in the IM to ensure successful anthesis in chrysanthemum.

The eukaryotic translation initiation factor eIF4E regulates flowering and circadian rhythm in Arabidopsis.

Translation initiation is a critical, rate-limiting step in protein synthesis. The eukaryotic translation initiation factor 4E (eIF4E) plays an essential role in this process. However, the mechanisms by which eIF4E-dependent translation initiation regulates plant growth and development remain not fully understood. In this study, we found that Arabidopsis eIF4E proteins are distributed in both the nucleus and cytoplasm, with only the cytoplasmic eIF4E being involved in the control of photoperiodic flowering. Genome-wide translation profiling using Ribo-tag sequencing reveals that eIF4E may regulate plant flowering by maintaining the homeostatic translation of components in the photoperiodic flowering pathway. eIF4E not only regulates the translation of flowering genes such as FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FLD) but also influences the translation of circadian genes like CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and PSEUDO-RESPONSE REGULATOR 9 (PRR9). Consistently, our results show that the eIF4E modulates the rhythmic oscillation of the circadian clock. Together, our study provides mechanistic insights into how the protein translation regulates multiple developmental processes in Arabidopsis, including the circadian clock and photoperiodic flowering.

Plant Circadian Clocks: Unravelling the Molecular Rhythms of Nature

The circadian clock is a fundamental biological mechanism that allows organisms to synchronize their internal processes with the external environment, thereby optimizing growth, development, and physiology. In plants, circadian rhythms govern various aspects of their life cycle, including germination, leaf movement, flowering, and responses to environmental cues such as light and temperature. Understanding the molecular mechanisms underlying plant circadian clocks is essential not only for elucidating fundamental principles of plant biology but also for applications in agriculture and biotechnology. This review delves into the intricate molecular networks that comprise plant circadian clocks, focusing on key components and their interactions. At the core of these clocks are transcription-translation feedback loops (TTFLs) involving a set of clock genes. The central oscillator, composed of genes such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), and PSEUDO-RESPONSE REGULATOR (PRR) genes, drives rhythmic expression of downstream clock-controlled genes (CCGs). The interplay between positive and negative regulators within the TTFLs generates robust oscillations with a period of approximately 24 hours. Additionally, post-translational modifications, protein-protein interactions, and chromatin remodeling contribute to fine-tuning the circadian system, allowing plants to adapt to changing environmental conditions. Light perception through photoreceptors such as phytochromes and cryptochromes plays a crucial role in entraining the circadian clock to external cues, ensuring synchronization with the day-night cycle. Furthermore, recent advancements in omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, have provided unprecedented insights into the complexity and plasticity of plant circadian clocks. Integration of multi-omics data has facilitated the construction of comprehensive regulatory networks and computational models, enabling predictive understanding of clock function and behavior under diverse conditions. Overall, unraveling the molecular rhythms of plant circadian clocks not only enhances our knowledge of fundamental biological processes but also holds promise for improving crop productivity, stress resilience, and sustainability in agriculture through targeted manipulation of clock components and their associated pathways. Future research endeavors will undoubtedly continue to unveil the intricacies of these fascinating timekeeping mechanisms, further enriching our understanding of the molecular rhythms of nature.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDO RESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its transcriptional repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 protein activity is unknown. We used double mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR (CBF) and COLD REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. We establish that HOS15 mediates PRR7 protein turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoter regions of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated regulation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways leading to freezing tolerance through upregulation of CBF1 and COR15A.

Genome-editing of a circadian clock gene TaPRR95 facilitates wheat peduncle growth and heading date

Plant height and heading date are important agronomic traits in wheat (Triticum aestivum L.) that affect final grain yield. In wheat, knowledge of pseudo-response regulator (PRR) genes on agronomic traits is limited. Here, we identify a wheat TaPRR95 gene by genome-wide association study to be associated with plant height. Triple allele mutant plants produced by CRISPR/Cas9 show increased plant height, particularly the peduncle, with an earlier heading date. The longer peduncle is mainly caused by the increased cell elongation at its upper section, whilst the early heading date is accompanied by elevated expression of flowering genes, such as TaFT and TaCO1. A peduncle-specific transcriptome analysis reveals up-regulated photosynthesis genes and down-regulated IAA/Aux genes for auxin signaling in prr95aabbdd plants that may act as a regulatory mechanism to promote robust plant growth. A haplotype analysis identifies a TaPRR95-B haplotype (Hap2) to be closely associated with reduced plant height and increased thousand-grain weight. Moreover, the Hap2 frequency is higher in cultivars than that in landraces, suggesting the artificial selection on the allele during wheat breeding. These findings suggest that TaPRR95 is a regulator for plant height and heading date, thereby providing an important target for wheat yield improvement.

Development and validation of a functional molecular marker for a CaAPRR2-like gene that controls green fruit colour in pepper

Fruit colour is a crucial factor influencing both the marketability and quality of pepper (Capsicum annuum), particularly ornamental varieties. Fruit colour is a complex multigenic trait in plant species. Previously, the Arabidopsis pseudo-response regulator 2 (APRR2) gene, one of the regulators that control fruit chlorophyll content and chloroplast development, thereby influencing fruit colour at the green immature stage in tomato (Solanum lycopersicum), cucumber (Cucumis sativus) and pepper, was reported. Functional molecular markers associated with this homologous gene in pepper could be employed to discern fruit colour at the seedling stage, thus improving the efficiency of green/white-fruited pepper breeding. In this study, a derived cleaved amplified polymorphic sequences (dCAPS) marker was developed based on the mutation site in the sequence of a CaAPRR2-like gene that was cloned from the inbred line B1-2, which exhibits milky white fruit at the green immature stage. The marker was subsequently validated in a CaAPRR2-like progeny segregation population. After digestion with the restriction endonuclease TaqI, the amplification product exhibited evident polymorphic bands, enabling it to distinguish between peppers with milky white and green fruit colours. The developed molecular marker displayed remarkable stability and repeatability, thus offering a straightforward and effective tool for enhancing fruit colour in pepper breeding programs.

PSEUDO-RESPONSE REGULATOR 3b and transcription factor ABF3 modulate abscisic acid-dependent drought stress response in soybean.

The circadian system plays a pivotal role in facilitating the ability of crop plants to respond and adapt to fluctuations in their immediate environment effectively. Despite the increasing comprehension of PSEUDO-RESPONSE REGULATORs and their involvement in the regulation of diverse biological processes, including circadian rhythms, photoperiodic control of flowering, and responses to abiotic stress, the transcriptional networks associated with these factors in soybean (Glycine max (L.) Merr.) remain incompletely characterized. In this study, we provide empirical evidence highlighting the significance of GmPRR3b as a crucial mediator in regulating the circadian clock, drought stress response, and abscisic acid (ABA) signaling pathway in soybeans. A comprehensive analysis of DNA affinity purification sequencing and transcriptome data identified 795 putative target genes directly regulated by GmPRR3b. Among them, a total of 570 exhibited a significant correlation with the response to drought, and eight genes were involved in both the biosynthesis and signaling pathways of ABA. Notably, GmPRR3b played a pivotal role in the negative regulation of the drought response in soybeans by suppressing the expression of abscisic acid-responsive element-binding factor 3 (GmABF3). Additionally, the overexpression of GmABF3 exhibited an increased ability to tolerate drought conditions, and it also restored the hypersensitive phenotype of the GmPRR3b overexpressor. Consistently, studies on the manipulation of GmPRR3b gene expression and genome editing in plants revealed contrasting reactions to drought stress. The findings of our study collectively provide compelling evidence that emphasizes the significant contribution of the GmPRR3b-GmABF3 module in enhancing drought tolerance in soybean plants. Moreover, the transcriptional network of GmPRR3b provides valuable insights into the intricate interactions between this gene and the fundamental biological processes associated with plant adaptation to diverse environmental conditions.

BoPRR9, a pseudo-response regulator protein from cabbage, plays a negative regulatory role in the response to cold stress

Cold stress can adversely affect the growth, development, and yield of plants. Exploring the cabbage cold-stress-related genes and their regulatory mechanisms is crucial for cultivating high-quality varieties of cabbage. The PSEUDO RESPONSE REGULATOR (PRR) factor plays an important role in plant response to abiotic stress. However, cabbage BoPRRs in cold tolerance are rarely reported. In this study, the PRR family gene BoPRR9 was isolated from cabbage (Brassica oleracea var. capitata L) and functionally characterized in transgenic Arabidopsis and cabbage in cold stress response. BoPRR9 encodes a protein located in the nucleus. The gene was strongly induced by cold treatment, and the transcription level of BoPRR9 was up regulated under cold stress. Under cold stress, overexpression of BoPRR9 resulted in enhanced cold sensitivity in transgenic Arabidopsis and cabbage, lower antioxidant enzyme activity and maximum photosynthetic efficiency of photosystem II(Fv/Fm), and higher relative electrolyte leakage (REL) and malondialdehyde (MDA) content. Meanwhile, the ability of the transgenic plants to remove reactive oxygen species (ROS) was significantly reduced. Additionally, the expression levels of cold-related genes were downregulated in BoPRR9-overexpressing plants. The results showed that the BoPRR9 gene negatively regulates cold stress, which provides clues for further understanding of the molecular mechanism of PRR-mediated cold stress response.

Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

Loss of daylength sensitivity by splice site mutation in Cannabis pseudo-response regulator.

Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for short day (SD) flowering requirements making outdoor cultivation in high latitudes possible. However, the benefits of photoperiod insensitivity are counterbalanced by low cannabinoid content and poor flower quality in auto-flowering genotypes. Despite recent studies in cannabis flowering, a mechanistic understanding of photoperiod insensitivity is still lacking. We used a combination of genome-wide association study and genetic fine-mapping to identify the genetic cause of auto-flowering in cannabis. We then used gene expression analyses and transient transformation assays to characterize flowering time control. Herein, we identify a splice site mutation within circadian clock gene PSEUDO-RESPONSE REGULATOR 37 (CsPRR37) in auto-flowering cannabis. We show that CsPRR37 represses FT expression and its circadian oscillations transition to a less repressive state during SD as compared to long days (LD). We identify several key circadian clock genes whose expression is altered in auto-flowering cannabis, particularly under non-inductive LD. Research into the pervasiveness of this mutation and others affecting flowering time will help elucidate cannabis domestication history and advance cannabis breeding toward a more sustainable outdoor cultivation system.

Potential locus W and candidate gene McPRR2 associated with pericarp pigment accumulation in bitter gourd (Momordica charantia L.) revealed via BSA-seq analysis

Pericarp color is a prominent agronomic trait that exerts a significant impact on consumer and breeder preferences. Genetic analysis has revealed that the pericarp color of bitter gourd is a quantitative trait. However, the underlying mechanism for this trait in bitter gourd remains largely unknown. In the present study, we employed bulked segregant analysis (BSA) to identify the candidate genes responsible for bitter gourd pericarp color (specifically, dark green versus white) within F2 segregation populations resulting from the crossing of B07 (dark green pericarp) and A06 (white pericarp). Through genomic variation, genetic mapping, and expression analysis, we identified a candidate gene named McPRR2, which was a homolog of Arabidopsis pseudo response regulator 2 (APRR2) encoded by LOC111023472. Sequence alignment of the candidate gene between the two parental lines revealed a 15-bp nucleotide insertion in the coding region of LOC111023472, leading to a premature stop codon and potentially causing a loss-of-function mutation. qRT-PCR analysis demonstrated that the expression of McPRR2 was significantly higher in B07 compared to A06, and it was primarily expressed in the immature fruit pericarp. Moreover, overexpression of McPRR2 in tomato could enhance the green color of immature fruit pericarp by increasing the chlorophyll content. Consequently, McPRR2 emerged as a strong candidate gene regulating the bitter gourd pericarp color by influencing chlorophyll accumulation. Finally, we developed a molecular marker linked to pericarp color, enabling the identification of genotypes in breeding populations. These findings provided valuable insights into the genetic improvement of bitter gourd pericarp color.

Genome-wide identification and expression profiling of Pseudo-Response Regulator (PRR) gene family in tomato

Circadian clocks are intrinsic time-keeping mechanisms that facilitate the coordination of development and physiological processes in plants with seasonal and diurnal variations in the environment. Pseudo-response regulator (PRR) genes regulate various essential processes, such as development, flowering time, and the formation of inflorescence architecture in the reproductive stage, principally by regulating transcriptional activity and protein function. The present research focused on genome-wide characterization, phylogenetic, cis-regulatory elements, miRNAs, 3D-sturcutural and expression profiling of the PRR proteins in tomato. Overall, 13 candidate genes were identified on six chromosomes (Chr1, Chr3, Chr4, Chr6, Chr8 and Chr10). Phylogenetic analysis categorized SlPRR gene members into four groups. All genes possess conserved PRR domains with unique sequences within these domains. Additionally, SlPRR genes exhibited diversity in numbers of exons and introns indicating potential functional divergence. Through comparative synteny investigation, it was ascertained that duplication events occurred. Furthermore, miRNAs were predicted to understand post-transcriptional changes in SlPRR genes. The results showed that SlPRR6 and SlPRR13 were targeted by 6 miRNAs each, while SlPRR1 and SlPRR3 were only targeted by 3 miRNAs. Three-dimensional (3D) configurations of 13 PRR proteins were also predicted. PRR genes exhibited differential expression pattern at both vegetative and reproductive stages in different time intervals. This pattern follows the sequence of early morning genes exhibiting the highest expression, followed by evening, and then noon. This implies that SlPRR genes have significant functions in various photoperiod signaling pathways throughout distinct periods during vegetative and reproductive stages. These findings stipulate a comprehensive understanding of the tomato PRR which can be employed for future functional studies of tomato PRR family members and their involvement in the intricate network of the circadian rhythm for regulating growth and development of plants.

Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

Flowering time regulator qFT13-3 involved in soybean adaptation to high latitudes.

Soybean is a short-day plant that typically flowers earlier when exposed to short-day conditions. However, the identification of genes associated with earlier flowering time but without a yield penalty is rare. In this study, we conducted genome-wide association studies (GWAS) using two re-sequencing datasets that included 113 wild soybeans (G. soja) and 1192 cultivated soybeans (G. max), respectively, and simultaneously identified a candidate flowering gene, qFT13-3, which encodes a protein homologous to the pseudo-response regulator (PRR) transcription factor. We identified four major haplotypes of qFT13-3 in the natural population, with haplotype H4 (qFT13-3H4) being lost during domestication, while qFT13-3H1 underwent natural and artificial selection, increasing in proportion from 4.5% in G. soja to 43.8% in landrace and to 81.9% in improve cultivars. Notably, most cultivars harbouring qFT13-3H1 were located in high-latitude regions. Knockout of qFT13-3 accelerated flowering and maturity time under long-day conditions, indicating that qFT13-3 functions as a flowering inhibitor. Our results also showed that qFT13-3 directly downregulates the expression of GmELF3b-2 which is a component of the circadian clock evening complex. Field trials revealed that the qft13-3 mutants shorten the maturity period by 11 days without a concomitant penalty on yield. Collectively, qFT13-3 can be utilized for the breeding of high-yield cultivars with a short maturity time suitable for high latitudes.

The Roles of the PSEUDO-RESPONSE REGULATORs in Circadian Clock and Flowering Time in Medicago truncatula

PSEUDO-RESPONSE REGULATORs (PRRs) play key roles in the circadian rhythms and flowering in plants. Here, we identified the four members of the PRR family in Medicago truncatula, including MtPRR9a, MtPRR9b, MtPRR7 and MtPRR5, and isolated their Tnt1 retrotransposon-tagged mutants. They were expressed in different organs and were nuclear-localized. The four MtPRRs genes played important roles in normal clock rhythmicity maintenance by negatively regulating the expression of MtGI and MtLHY. Surprisingly, the four MtPRRs functioned redundantly in regulating flowering time under long-day conditions, and the quadruple mutant flowered earlier. Moreover, MtPRR can recruit the MtTPL/MtTPR corepressors and the other MtPRRs to form heterodimers to constitute the core mechanism of the circadian oscillator.