Live cell imaging reveals plant aurora kinase has dual roles during mitosis

Abstract
The meticulous and precise segregation of chromosomes during the intricate process of mitosis stands as an absolutely fundamental requirement for the accurate and equitable distribution of an organism’s complete genetic information to the two newly formed daughter cells. This exactitude is paramount for maintaining genomic stability across generations of cells and is essential for the healthy growth, development, and propagation of all eukaryotic life forms. Errors in this highly orchestrated process, even minor ones, can lead to aneuploidy, a condition where cells possess an abnormal number of chromosomes, which is frequently associated with developmental disorders, cellular dysfunction, and various diseases, including cancer in animals and detrimental effects on plant viability and vigor. Therefore, understanding the molecular mechanisms that govern this precision is of immense scientific importance.

In this comprehensive investigation, advanced live cell imaging techniques were strategically employed to meticulously observe the dynamic behavior of both microtubules and kinetochores within living plant cells. The experiments were specifically conducted using tobacco BY-2 cells, a well-established and highly advantageous model system in plant cell biology renowned for its rapid and synchronized division, making it ideal for studying cell cycle events. The primary experimental intervention involved treating these cells with hesperadin, a potent and specific pharmacological inhibitor of Aurora kinase. By inhibiting the activity of this crucial enzyme, the study aimed to dissect and elucidate the precise functional roles that plant Aurora kinase plays throughout the complex stages of mitosis.

The observational analysis of the cellular responses to hesperadin treatment yielded significant and informative results. A particularly notable finding was that the inhibition of Aurora kinase activity led directly to a pronounced delay in the proper alignment of CenH3, a critical centromeric histone H3 variant that serves as a molecular marker for kinetochores, on the precise plane of the spindle equator. This delay indicates a disruption in the timely progression through metaphase, suggesting that plant Aurora kinase is critically involved in the precise positioning and stable attachment of chromosomes to the mitotic spindle. Furthermore, as the process of chromosome segregation commenced and progressed into anaphase, detailed live cell imaging revealed the presence of what are known as lagging CenH3s, which represent chromosomes that fail to move synchronously or correctly to the spindle poles. Crucially, two distinct types of dynamic behaviors were observed for these lagging CenH3s. The very presence of such distinct dynamics underscores the complexity of the errors induced by Aurora kinase inhibition and points to multiple pathways or checkpoints being affected by its absence. These observations collectively indicate a multifaceted role for the plant Aurora kinase in ensuring the integrity of the mitotic process.

The cumulative findings from this in-depth study compellingly indicate that the plant Aurora kinase possesses a sophisticated and dualistic regulatory capacity within the context of mitotic cell division. Its first crucial role involves the vigilant correction of aberrant kinetochore–microtubule attachments. During metaphase, each sister kinetochore must attach to microtubules originating from opposite spindle poles, a configuration known as amphitelic attachment. Misattachments, such as syntelic (both sister kinetochores attach to microtubules from the same pole) or merotelic (a single kinetochore attaches to microtubules from both poles), are detrimental and must be corrected to prevent aneuploidy. Aurora kinase is known to localize to kinetochores and spindle microtubules, where its phosphorylation activity helps to destabilize incorrect attachments, allowing for their detachment and subsequent re-attachment in a proper configuration. This proofreading mechanism is absolutely essential for ensuring that every chromosome is correctly aligned and under appropriate tension before anaphase onset.

The second equally vital function attributed to the plant Aurora kinase is its integral involvement in the dissociation of cohesin. Cohesin is a protein complex that physically links sister chromatids together, holding them as a single unit from their replication during S phase until anaphase. For sister chromatids to separate and migrate to opposite poles, this cohesin complex must be efficiently and timely removed or cleaved. The findings suggest that plant Aurora kinase plays a direct or indirect role in facilitating this crucial cohesin dissociation, a process that is prerequisite for the successful segregation of chromosomes. This involvement spans both the period of chromosome alignment on the metaphase plate and the subsequent active segregation phase. By participating in both the refinement of kinetochore–microtubule interactions and the timely release of sister chromatids, plant Aurora kinase emerges as an indispensable orchestrator of chromosome fidelity, ensuring the accurate transmission of genetic material in plant cells.

Keywords
Chromosome segregation; Cohesin dissociation; Kinetochore–microtubule attachment; Lagging chromosome; Plant Aurora kinase.

Introduction
The precise and accurate segregation of chromosomes during the pivotal process of mitosis is an absolutely critical cellular event, as any deviation can lead to genetic anomalies. Errors in this complex choreography directly result in aneuploidy, a state characterized by an abnormal number of chromosomes within a cell. This condition, where there is a net loss or gain of one or more individual chromosomes, fundamentally disrupts the delicate balance of gene dosage, leading to imbalances in the expression levels of genes located on the affected chromosomes. Such gene dosage alterations can have profound physiological consequences, as many cellular processes are exquisitely sensitive to the precise quantity of genetic material present.

While it is generally observed that plants tend to exhibit a greater tolerance to the presence of extra chromosomes compared to animals, even in the plant kingdom, aneuploid genomes are not entirely stable and can still lead to deleterious effects on growth, development, and viability. Aneuploidy can arise from a multitude of cellular defects, underscoring the complexity and numerous checkpoints involved in ensuring genomic integrity. These defects can include issues in the duplication, maturation, or segregation of centrosomes, which serve as the primary microtubule-organizing centers in many eukaryotic cells. Additionally, problems with chromosome cohesion, where sister chromatids fail to remain properly linked, can contribute to aneuploidy.

Malfunctions in the spindle assembly checkpoint, a critical surveillance mechanism that ensures all chromosomes are properly attached to the mitotic spindle before anaphase initiation, are also significant contributors. Furthermore, improper attachment of chromosomes to the spindle microtubules themselves represents a direct and common pathway to segregation errors. Among the key regulatory proteins involved in ensuring proper chromosome-microtubule attachment, Aurora B kinase has been extensively studied. Indeed, experimental inactivation of Aurora B through various methodologies in mammalian cells has been shown to induce a spectrum of severe chromosome segregation defects, notably including the pervasive presence of lagging chromosomes, which are chromosomes that fail to move correctly to the spindle poles during anaphase.

In the realm of plant biology, homologous Aurora kinase genes have been identified and characterized. Specifically, in the model plant *Arabidopsis thaliana*, three distinct Aurora kinase genes—designated AtAUR1, AtAUR2, and AtAUR3—have been elucidated. Research has confirmed that all three AtAUR isoforms possess the enzymatic capability to phosphorylate histone H3 at serine 10 in vitro, a modification known to be intimately associated with chromosome condensation and mitotic progression. Furthermore, specific studies have indicated that AtAUR2 might also play a role in regulating cell division through a more complex mechanism involving competition with an AtAUR2 splicing variant, suggesting an additional layer of regulatory control. To investigate the functional significance of plant Aurora kinase in a living cellular context, the well-established tobacco BY-2 cell line (*Nicotiana tabacum* cv. Bright Yellow-2) has proven invaluable. Treatment of these cells with hesperadin, a potent chemical inhibitor of Aurora kinase, was demonstrated to effectively suppress the phosphorylation of histone H3 at both serine 10 and serine 28, providing strong evidence that hesperadin indeed inhibits the activity of plant Aurora kinase within these cells.

Critically, this inhibition was correlated with the induction of widespread aberrant chromosome segregation, manifesting as the frequent formation of lagging chromosomes and the appearance of micronuclei, which are small nuclei containing mis-segregated chromosomes. Despite these significant observations from fixed cell studies, the precise mechanistic details underlying the formation of lagging chromosomes during chromosome segregation in plants, especially concerning the dynamic interplay of kinetochores and microtubules, had remained largely unexplored and unclear.

To address this knowledge gap and gain a deeper understanding of the dynamics of kinetochores and microtubules in living hesperadin-treated BY-2 cells, the researchers embarked on an ambitious project. They meticulously generated a transgenic tobacco BY-2 cell line, a powerful experimental tool that stably expresses specific fluorescently tagged proteins designed to visualize key mitotic structures. This engineered cell line expressed alpha-tubulin fused with green fluorescent protein (GFP-alpha-tubulin), enabling the real-time visualization of microtubules, the primary components of the mitotic spindle. Simultaneously, it expressed CenH3 (centromeric histone H3, specifically the *Arabidopsis* HTR12 homolog) fused with tdTomato, a tandem dimer variant of red fluorescent protein (RFP-CenH3). The RFP-CenH3 construct served as a robust marker for visualizing kinetochores, the protein complexes assembled on centromeres that mediate chromosome attachment to microtubules. This specially engineered cell line was designated as BY-GTRC (BY-2 cells stably expressing GFP-alpha-tubulin-RFP-CenH3).

With this innovative cell line in hand, researchers employed a sophisticated wide-field inverted microscope system for live cell observation. Their initial observations of unperturbed BY-GTRC cells provided a baseline understanding of normal mitotic progression. Following nuclear envelope breakdown (NEBD), a critical event marking the onset of prometaphase, kinetochore microtubules were seen to rapidly organize and establish connections. As the cells progressed into metaphase, the CenH3s, marking the kinetochores, exhibited characteristic oscillatory movements and swiftly aligned themselves precisely on the spindle equator, forming the metaphase plate. Upon the achievement of complete CenH3 alignment, a crucial checkpoint for proper segregation, the CenH3s were observed to equally separate and migrate to opposite poles, indicative of anaphase onset. Subsequently, the formation of phragmoplasts, structures essential for cell plate formation in plants, was observed during anaphase and telophase. These initial observations unequivocally confirmed that the BY-GTRC cells provided an excellent live imaging platform for real-time visualization and detailed analysis of kinetochore and microtubule dynamics throughout the entirety of mitosis.

With the baseline dynamics established, the core of the investigation focused on elucidating the specific effects of Aurora kinase inhibition on these dynamics. Live cell analysis was performed on BY-GTRC cells after they had been treated with hesperadin for 41 hours. Detailed observation of GFP-alpha-tubulin and RFP-CenH3 dynamics commenced immediately after NEBD. A significant finding was that in hesperadin-treated cells, the alignment of CenH3s on the spindle equator was notably delayed, occurring approximately 28 minutes after NEBD. In stark contrast, in the untreated control cells, this alignment was completed much earlier, at around 14 minutes after NEBD. Quantitatively, the mean time from NEBD to complete CenH3 alignment (CCA) in hesperadin-treated cells was 27.4 minutes, with a range extending from 19.0 to 52.0 minutes. This was a statistically significant prolongation compared to the control cells, which exhibited a mean time of 14.3 minutes with a narrower range of 8.0 to 19.0 minutes (P < 0.001, n = 10). This compelling result strongly indicated that Aurora kinase inhibition by hesperadin directly caused a substantial delay in the proper and timely alignment of CenH3s on the spindle equator.

Furthermore, a critical observation during anaphase in hesperadin-treated BY-GTRC cells was the consistent presence of lagging CenH3s, signifying mis-segregated chromosomes. Despite these significant delays and initial misalignments, it was noted that in many instances, the CenH3s in hesperadin-treated cells did eventually align on the spindle equator and proceed to segregate, although with altered dynamics. Interestingly, while the time from NEBD to complete CenH3 alignment was significantly extended, the overall duration from NEBD to metaphase itself did not show a statistically significant difference between hesperadin-treated cells (mean 36.8 min) and control cells (mean 45.4 min). This suggests a prolonged prometaphase or a slower process of attachment correction within the metaphase window. Importantly, the dynamics of the microtubules themselves, as observed via GFP-alpha-tubulin, did not appear to differ significantly between the control and hesperadin-treated cells. Similarly, the total time spent in mitosis was not significantly different between the two groups. These nuanced results collectively demonstrated that hesperadin specifically induced a delay in CenH3 alignment on the spindle equator and resulted in the formation of lagging CenH3s during the process of chromosome segregation, while the general progression and duration of other mitotic events remained largely normal.

To delve into the precise kinematic characteristics of CenH3 movements, a detailed manual tracking of RFP-CenH3 trajectories was performed in hesperadin-treated BY-GTRC cells. This meticulous analysis allowed for the measurement of the distances of individual CenH3s from the spindle equator over time. While CenH3s in hesperadin-treated cells still exhibited oscillations as they attempted to align on the spindle equator, the time required to achieve complete alignment (NEBD to CCA) was indeed increased compared to control cells. More importantly, the slopes of the graphs representing CenH3 movement in hesperadin-treated cells were notably shallower than those observed in control cells. This critical finding indicated that the oscillation velocity of CenH3s in hesperadin-treated cells was significantly slower than in untreated cells. Statistical analysis of the distribution of CenH3 oscillation velocities further confirmed this, revealing that the average velocity in control cells was 1.6 ± 1.0 micrometers per minute, whereas in hesperadin-treated cells, it was a substantially reduced 0.7 ± 0.4 micrometers per minute. This direct evidence confirmed that hesperadin, by inhibiting Aurora kinase, caused a marked decrease in the velocity of CenH3 movement towards the spindle equator during chromosome alignment in BY-GTRC cells.

The concept of bi-oriented kinetochore-microtubule attachment is paramount for the faithful segregation of chromosomes. However, during the dynamic process of chromosome alignment, two primary types of aberrant attachments can occur. In syntelic attachment, both sister kinetochores of a single chromosome become erroneously attached to microtubules originating from the *same* spindle pole. In contrast, merotelic attachment involves a scenario where one sister kinetochore is incorrectly attached to microtubules originating from *both* spindle poles. While syntelically attached chromosomes typically remain positioned abnormally close to one of the spindle poles due to the unopposed pulling forces, merotelically attached chromosomes can, remarkably, still align on the spindle equator. This is because, unlike syntelic attachments, merotelic attachments are technically "bi-oriented" in the sense that the chromosome is pulled by microtubules from both poles, albeit improperly. However, a key distinction is that merotelically attached chromosomes move towards the spindle equator with a significantly slower velocity compared to chromosomes that possess proper kinetochore-microtubule attachments. Given the observed reduction in CenH3 oscillation velocity and the delayed alignment in hesperadin-treated cells, these results strongly suggested that aberrant kinetochore-microtubule attachments, specifically merotelic attachments, were occurring during chromosome alignment when plant Aurora kinase was inhibited in BY-GTRC cells.

Interestingly, previous studies in mammalian cells have shown that Aurora kinase inhibition can induce syntelic attachments, often leading to pronounced chromosome misalignment. However, in the present study using hesperadin-treated BY-GTRC cells, the researchers did not observe chromosomes remaining persistently near the spindle poles, which would be characteristic of syntelic attachments. This difference might suggest a plant-specific response or a different threshold for inhibition. It is known that partial inhibition of Aurora kinase in mammalian cells can lead to lagging chromosomes in anaphase by increasing the prevalence of merotelic kinetochores. To further probe the effects of stronger inhibition, the dynamics of CenH3 were also observed in BY-GTRC cells treated with a significantly higher concentration of hesperadin (50 micromolar). Even at this elevated concentration, CenH3s were still observed to eventually align on the spindle equator, albeit with a prolonged time from NEBD to CCA compared to control cells. The consistent observation of lagging CenH3s in almost all hesperadin-treated BY-GTRC cells across different concentrations corroborated the widespread nature of this segregation defect. While the frequency of lagging chromosomes was high, a portion of these lagging CenH3s were observed to eventually move and become reincorporated into one of the daughter nuclei, indicating that not all segregation errors necessarily lead to micronucleus formation, suggesting a degree of plasticity or residual corrective mechanisms.

To gain even finer resolution into the mechanisms underlying lagging chromosome formation, detailed manual tracking of individual CenH3s was performed specifically during the anaphase and telophase stages in hesperadin-treated BY-GTRC cells. This meticulous analysis revealed two distinct types of dynamic behaviors for lagging CenH3s during chromosome segregation. The first type of lagging behavior was characterized by a scenario where one CenH3 of a chromosome exhibited dynamics similar to a normally segregating chromosome, while its sister CenH3 was markedly lagged during anaphase. This particular lagging CenH3 was also observed to be visibly stretched on the spindle equator, indicative of tension and pulling forces from both poles. Remarkably, this stretched CenH3 was subsequently observed to separate into three distinct parts, a highly unusual event. This specific observation strongly suggests that Aurora kinase inhibition induced merotelic attachment in the BY-GTRC cells. In such a scenario, if a syntelic attachment were to initially occur, the subsequent capture of microtubules from the opposite pole by the already syntelically attached kinetochore would result in a merotelic attachment. This transition could explain the observed delay in complete CenH3 alignment. During anaphase, a merotelic kinetochore would experience persistent pulling forces from both poles, leading to the observed stretching and eventual fragmentation.

The second distinct type of lagging behavior observed involved both CenH3s of a particular chromosome. In this instance, both CenH3s remained persistently on the spindle equator even at the very onset of anaphase, only separating much later than their counterparts. This observation provides compelling evidence that hesperadin, by inhibiting Aurora kinase, also induced an inhibition of cohesin dissociation specifically on these affected chromosomes. Sister chromatid separation, the hallmark event of anaphase, is absolutely dependent on the timely and complete loss of cohesion between sister chromatids, a process that precisely coincides with the proteolytic dissociation of the cohesin complex. In vertebrate cells, cohesin removal is known to occur via two distinct pathways. A significant portion of cohesin is removed from chromosome arms during prophase, in what is known as the "prophase pathway," a process that requires the activity of Polo-like kinase and Aurora B. Subsequently, once all chromosomes are properly bi-oriented on the metaphase plate, the centromeric cohesin is cleaved by the protease separase, triggering anaphase onset. In plants, the *Arabidopsis* separase AESP is known to be essential for cohesin removal during meiosis. Therefore, the observation that both CenH3s of a chromosome remained lagged on the equator strongly implies that plant Aurora kinase plays a crucial and previously underappreciated role in facilitating the proper and timely removal of cohesin from chromosomes during plant mitosis.

In conclusion, the application of sophisticated live cell imaging of kinetochores and microtubules in the meticulously engineered BY-GTRC cells has provided profound new insights, revealing that plant Aurora kinase performs crucial dual roles during the complex processes of chromosome alignment and segregation.

The administration of hesperadin, an inhibitor of Aurora kinase, demonstrably induced a significant delay in the alignment of CenH3s on the spindle equator, primarily by substantially slowing their oscillation velocity. Furthermore, the observation of stretched lagging CenH3s during anaphase in the hesperadin-treated cells strongly suggests that inhibition of plant Aurora kinase leads to the formation of aberrant kinetochore-microtubule attachments, specifically merotelic attachments. Thus, it is now proposed that plant Aurora kinase is a key regulator of kinetochore-microtubule attachments, actively participating in the quality control and correction mechanisms during chromosome alignment.

In addition to this critical role, the study also provided clear evidence that plant Aurora kinase is intimately involved in the essential process of cohesin dissociation during chromosome segregation. This was evidenced by the persistent lagging of both CenH3s of a chromosome on the spindle equator in hesperadin-treated BY-GTRC cells, indicating a failure of sister chromatid separation due to impaired cohesin removal. While previous studies using fixed cells had reported a general function for plant Aurora kinase in chromosome segregation, the unparalleled resolution and dynamic capabilities afforded by live cell imaging, coupled with the visualization of kinetochores and microtubules, have enabled the precise identification and elucidation of these distinct and fundamental roles for plant Aurora kinase in ensuring the fidelity of chromosome alignment and subsequent segregation in plant mitotic division.

Materials and Methods
The experimental procedures commenced with the meticulous maintenance of tobacco BY-2 cells, a cell line widely recognized and utilized as an excellent model system for plant cell division studies. These cells were cultured precisely according to established protocols previously described by Nagata and colleagues in 1992, ensuring consistency and reproducibility of the starting biological material. The BY-2 cells were sustained in a modified Linsmaier and Skoog medium, a nutrient-rich formulation optimized for plant cell proliferation, and were continuously agitated in a rotary shaker to ensure adequate aeration and uniform nutrient distribution. The culturing conditions were strictly controlled, with the cells maintained at a constant temperature of 26 degrees Celsius in complete darkness, fostering optimal growth and synchronized cell division.

For the purpose of live cell imaging and the visualization of key mitotic structures, specific expression vectors were engineered. The initial step involved the construction of an RFP (red fluorescent protein) expression vector. This was achieved by systematically replacing the GFP (green fluorescent protein) coding sequence within the pre-existing spUC-GFP vector, as detailed by Fujimoto et al. in 2004. The GFP sequence was precisely substituted with a TA-cloned tdTomato sequence, a tandem dimer variant of RFP, originally sourced from the pCR2.1 vector supplied by Invitrogen, located in Carlsbad, California, USA. This newly constructed vector was subsequently renamed spUC-tdTomato. Following this, the RFP-CenH3 expression vector was meticulously designed and assembled. This involved the insertion of the TA-cloned *Arabidopsis thaliana* CenH3 gene (specifically the HTR12 isoform, annotated as At1g01370) from its pCR2.1 vector into the previously constructed spUC-tdTomato vector. To enable stable integration into the plant genome, the Sse8387I-digested spUC-tdTomato-CenH3 construct was then ligated with the PstI-digested binary vector pMDC99, a commonly used vector for plant transformation as described by Curtis and Grossniklaus in 2003.

The assembled binary vector, designated 35S::RFP-CenH3, was then introduced into *Agrobacterium tumefaciens* strain EHA101, a bacterial vehicle frequently employed for transferring genetic material into plant cells. The subsequent transformation of BY-GT16 cells, which were a pre-existing transgenic BY-2 cell line stably expressing GFP-fused alpha-tubulin (as reported by Kumagai et al. in 2001), with the RFP-CenH3 construct was performed using a method akin to that described by Fujimoto et al. in 2004. A critical modification to the standard protocol involved the rigorous selection of transformants. This selection process was carried out using a triple antibiotic regimen, incorporating 500 milligrams per liter of claforan, 50 milligrams per liter of kanamycin, and 30 milligrams per liter of hygromycin, ensuring that only cells successfully incorporating both GFP-alpha-tubulin and RFP-CenH3 transgenes could survive and proliferate. The resultant double-transgenic cell line, possessing stable expression of both GFP-alpha-tubulin and RFP-CenH3, was formally designated as BY-GTRC, representing a novel and powerful tool for the specific visualization of microtubules and kinetochores in living plant cells.

For experimental treatments, hesperadin, a potent Aurora kinase inhibitor obtained from Boehringer Ingelheim Austria, Vienna, Austria, was prepared at two distinct concentrations: 5 micromolar and 50 micromolar. For control experiments, an equivalent volume of dimethylsulfoxide (DMSO), the solvent for hesperadin, was added to the cell cultures. These solutions were introduced to 3-day-old BY-GTRC cells, and the cells were subsequently cultured for an additional 1 hour following the addition of the inhibitor or control solvent. Following this incubation period, the treated BY-GTRC cells were carefully transferred into specialized Petri dishes. These dishes were equipped with poly-L-lysine-coated coverslips at the bottom, a treatment designed to promote cell adhesion and prevent movement during live imaging. These prepared dishes were then precisely positioned on the inverted platform of a high-resolution fluorescence microscope, specifically an Olympus IX-81 model. The microscope system was further equipped with a cooled charged-coupled device (CCD) camera, a CoolSNAP HQ2 from Roper Scientific, Houston, Texas, USA, which allowed for the acquisition of high-quality, low-noise images. Images were systematically acquired at regular intervals, specifically every 30 seconds, using a high-magnification 40x objective lens (UApo/340, numerical aperture 1.35, oil immersion), ensuring detailed capture of cellular dynamics. The subsequent processing and analysis of the acquired images were performed using a suite of specialized software, including MetaMorph from Universal Imaging Corporation, Downington, Pennsylvania, USA; WCIF ImageJ software, a public domain image processing program accessible at www.uhnresearch.ca/facilities/wcif/imagej/; and Adobe Photoshop CS3 Extended from Adobe Systems, San Jose, California, USA, for image enhancement and presentation.

Statistical analyses of the collected quantitative data were rigorously performed using GraphPad Prism version 5.01 for Windows, a comprehensive statistical software package from GraphPad Software, San Diego, California, USA. Specifically, two-way analysis of variance (ANOVA) coupled with a Bonferroni post hoc test was employed to determine statistically significant differences between experimental groups, ensuring the robustness and reliability of the conclusions drawn from the data. To analyze the intricate movements of individual RFP-CenH3 particles within the BY-GTRC cells, the Manual Tracking plug-in of the WCIF ImageJ software was utilized. This involved a meticulous process where researchers manually clicked on the centroid of each CenH3 signal across consecutive time-lapse images, thereby generating precise trajectories of their movements. Following this manual tracking, the distances of the CenH3s from the spindle equator were calculated with high precision using Microsoft Excel. For this calculation, the spindle equator was consistently defined as the exact position where CenH3s were perfectly aligned at the moment of complete CenH3 alignment, serving as a stable reference point. A unidirectional movement of 2 micrometers was established as the criterion for judging legitimate CenH3 movement, distinguishing purposeful migration from minor fluctuations or imaging noise.

Supplementary data pertinent to this study, providing additional details and supporting information, are made available and can be accessed online through the Plant and Cell Physiology journal platform.

Funding for this research was generously provided by several distinguished organizations. The Japan Society for the Promotion of Science for Young Scientists awarded funding to D.K. The Ministry of Education, Culture, Sports, Science and Technology of Japan provided Grant-in-Aid for Scientific Research, specifically under grant numbers 20370027 and 20061020, to S.M. Additionally, the Japan Science and Technology Agency contributed funding through its BIRD program to S.M. and its SENTAN program to K.F., collectively supporting the extensive research efforts.

The authors extend their sincere acknowledgments to several individuals and organizations whose contributions were invaluable to the success of this study. Special gratitude is extended to S. Hasezawa for generously providing the BY-GT16 cell line, which served as the foundational transgenic line. R.Y. Tsien is acknowledged for the provision of the tdTomato vector, a crucial component for the fluorescent labeling. N. Kraut is thanked for supplying the hesperadin, the key Aurora kinase inhibitor used in this investigation. The *Arabidopsis* Biological Resource Center is acknowledged for providing the pMDC99 binary vector. A. Kawabe is recognized for their expertise in cloning the HTR12 gene. Finally, R. Isobe is thanked for their invaluable technical assistance throughout the experimental work, contributing significantly to the execution and success of the research.