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Leaf senescence, the final stage of leaf development, is influenced by numerous internal and environmental signals. However, how biotic stresses such as pathogen infection regulate leaf senescence remains largely unclear. In this study, we found that the premature leaf senescence in Arabidopsis caused by the soil-borne vascular fungus Verticillium dahliae was impaired by disruption of a protein elicitor from V. dahliae 1 named PevD1. Constitutive or inducible overexpression of PevD1 accelerated Arabidopsis leaf senescence. Interestingly, a senescence-associated NAC transcription factor, ORE1, was targeted by PevD1. PevD1 could interact with and stabilize ORE1 protein by disrupting its interaction with the RING-type ubiquitin E3 ligase NLA. Mutation of ORE1 suppressed the premature senescence caused by overexpressing PevD1, whereas overexpression of ORE1 or PevD1 led to enhanced ethylene production and thereby leaf senescence. We showed that ORE1 directly binds the promoter of ACS6 and promotes its expression for mediating PevD1-induced ethylene biosynthesis. Loss-of-function of ACSs could suppress V. dahliae-induced leaf senescence in ORE1-overexpressing plants. Furthermore, we found thatPevD1 also interacts with Gossypium hirsutum ORE1 (GhORE1) and that virus-induced gene silencing of GhORE1 delays V. dahliae-triggered leaf senescence in cotton, indicating a possibly conserved mechanism in plants. Taken together, these results suggest that V. dahliae induces leaf senescence by secreting the effector PevD1 to manipulate the ORE1-ACS6 cascade, providing new insights into biotic stress-induced senescence in plants., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

All living organisms are unavoidably exposed to various endogenous and environmental stresses that trigger potentially fatal DNA damage, including double-strand breaks (DSBs). Although a growing body of evidence indicates that DNA damage is one of the prime drivers of aging in animals, little is known regarding the importance of DNA damage and its repair on lifespan control in plants. We found that the level of DSBs increases but DNA repair efficiency decreases as Arabidopsis leaves age. Generation of DSBs by inducible expression of I-PpoI leads to premature senescence phenotypes. We examined the senescence phenotypes in the loss-of-function mutants for 13 key components of the DNA repair pathway and found that deficiency in ATAXIA TELANGIECTASIA MUTATED (ATM), the chief transducer of the DSB signal, results in premature senescence in Arabidopsis. ATM represses DSB-induced expression of senescence-associated genes, including the genes encoding the WRKY and NAC transcription factors, central components of the leaf senescence process, via modulation of histone lysine methylation. Our work highlights the significance of ATM in the control of leaf senescence and has significant implications for the conservation of aging mechanisms in animals and plants., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

Leaf senescence is an important developmental process involving orderly disassembly of macromolecules for relocating nutrients from leaves to other organs and is critical for plants' fitness. Leaf senescence is the response of an intricate integration of various environmental signals and leaf age information and involves a complex and highly regulated process with the coordinated actions of multiple pathways. Impressive progress has been made in understanding how senescence signals are perceived and processed, how the orderly degeneration process is regulated, how the senescence program interacts with environmental signals, and how senescence regulatory genes contribute to plant productivity and fitness. Employment of systems approaches using omics-based technologies and characterization of key regulators have been fruitful in providing newly emerging regulatory mechanisms. This review mainly discusses recent advances in systems understanding of leaf senescence from a molecular network dynamics perspective. Genetic strategies for improving the productivity and quality of crops are also described.

Plant roots provide structural support and absorb nutrients and water; therefore, their proper development and function are critical for plant survival. Extensive studies on the early stage of ontogenesis of the primary root have revealed that the root apical meristem (RAM) undergoes dynamic structural and organizational changes during early germination. Quiescent center (QC) cells, a group of slowly dividing cells at the center of the stem-cell niche, are vital for proper function and maintenance of the RAM. However, temporal aspects of molecular and cellular changes in QC cells and their regulatory mechanisms have not been well studied. In the present study, we investigated temporal changes in QC cell size, expression of QC cell-specific markers (WOX5 and QC25), and genotoxic tolerance and division rate of QC cells in the Arabidopsis primary root. Our data revealed the decreased size of QC cells and the decreased expression of the QC cell-specific markers with root age. We also found that QC cell division frequency increased with root age. Furthermore, our study provides evidence supporting the link between the transition of QC cells from a mitotically quiescent state to the frequently dividing state and the decrease in tolerance to genotoxic stress.

Plant leaves undergo a series of developmental changes from leaf primordium initiation through growth and maturation to senescence throughout their life span. Although the mechanisms underlying leaf senescence have been intensively elucidated, our knowledge of the interrelationship between early leaf development and senescence is still fragmentary. We isolated the oresara15-1Dominant (ore15-1D) mutant, which had an extended leaf longevity and an enlarged leaf size, from activation-tagged lines of Arabidopsis. Plasmid rescue identified that ORE15 encodes a PLANT A/T-RICH SEQUENCE- AND ZINC-BINDING PROTEIN family transcription factor. Phenotypes of ore15-1D and ore15-2, a loss-of-function mutant, were evaluated through physiological and anatomical analyses. Microarray, quantitative reverse transcription polymerase chain reaction, and chromatin immunoprecipitation as well as genetic analysis were employed to reveal the molecular mechanism of ORE15 in the regulation of leaf growth and senescence. ORE15 enhanced leaf growth by promoting the rate and duration of cell proliferation in the earlier stage and suppressed leaf senescence in the later stage by modulating the GROWTH-REGULATING FACTOR (GRF)/GRF-INTERACTING FACTOR regulatory pathway. Our study highlighted a molecular conjunction through ORE15 between growth and senescence, which are two temporally separate developmental processes during leaf life span., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Leaf senescence is regulated by diverse developmental and environmental factors to maximize plant fitness. The red to far-red light ratio (R:FR) detected by plant phytochromes is reduced under vegetation shade, thus initiating leaf senescence. However, the role of phytochromes in promoting leaf senescence under FR-enriched conditions is not fully understood. In this study, we investigated the role of phyA and phyB in regulating leaf senescence under FR in Arabidopsis thaliana (Arabidopsis). FR enrichment and intermittent FR pulses promoted the senescence of Arabidopsis leaves. Additionally, phyA and phyB mutants showed enhanced and repressed senescence phenotypes in FR, respectively, indicating that phyA and phyB antagonistically regulate FR-dependent leaf senescence. Transcriptomic analysis using phyA and phyB mutants in FR identified differentially expressed genes (DEGs) involved in leaf senescence-related processes, such as responses to light, phytohormones, temperature, photosynthesis and defense, showing opposite expression patterns in phyA and phyB mutants. These contrasting expression profiles of DEGs support the antagonism between phyA and phyB in FR-dependent leaf senescence. Among the genes showing antagonistic regulation, we confirmed that the expression of WRKY6, which encodes a senescence-associated transcription factor, was negatively and positively regulated by phyA and phyB, respectively. The wrky6 mutant showed a repressed senescence phenotype compared with the wild type in FR, indicating that WRKY6 plays a positive role in FR-dependent leaf senescence. Our results imply that antagonism between phyA and phyB is involved in fine-tuning leaf senescence under varying FR conditions in Arabidopsis., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The circadian clock coordinates the daily cyclic rhythm of numerous biological processes by regulating a large portion of the transcriptome. In animals, the circadian clock is involved in aging and senescence, and circadian disruption by mutations in clock genes frequently accelerates aging. Conversely, aging alters circadian rhythmicity, which causes age-associated physiological alterations. However, interactions between the circadian clock and aging have been rarely studied in plants. Here, we investigated potential roles for the circadian clock in the regulation of leaf senescence in plants. Members of the evening complex in Arabidopsis circadian clock, EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), and LUX ARRHYTHMO (LUX), as well as the morning component PSEUDO-RESPONSE REGULATOR 9 (PRR9), affect both age-dependent and dark-induced leaf senescence. The circadian clock regulates the expression of several senescence-related transcription factors. In particular, PRR9 binds directly to the promoter of the positive aging regulator ORESARA1 ( ORE1 ) gene to promote its expression. PRR9 also represses miR164 , a posttranscriptional repressor of ORE1 Consistently, genetic analysis revealed that delayed leaf senescence of a prr9 mutant was rescued by ORE1 overexpression. Thus, PRR9, a core circadian component, is a key regulator of leaf senescence via positive regulation of ORE1 through a feed-forward pathway involving posttranscriptional regulation by miR164 and direct transcriptional regulation. Our results indicate that, in plants, the circadian clock and leaf senescence are intimately interwoven as are the clock and aging in animals., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

Leaf senescence involves degenerative but active biological processes that require balanced regulation of pro- and anti-senescing activities. Ethylene and cytokinin are major antagonistic regulatory hormones that control the timing and progression rate of leaf senescence. To identify the roles of these hormones in the regulation of leaf senescence in Arabidopsis, global gene expression profiles in detached leaves of the wild type, an ethylene-insensitive mutant (ein2/ore3), and a constitutive cytokinin response mutant (ahk3/ore12) were investigated during dark-induced leaf senescence. Comparative transcriptome analyses revealed that genes involved in oxidative or salt stress response were preferentially altered in the ein2/ore3 mutant, whereas genes involved in ribosome biogenesis were affected in the ahk3/ore12 mutant during dark-induced leaf senescence. Similar results were also obtained for developmental senescence. Through extensive molecular and physiological analyses in ein2/ore3 and ahk3/ore12 during dark-induced leaf senescence, together with responses when treated with cytokinin and ethylene inhibitor, we conclude that ethylene acts as a senescence-promoting factor via the transcriptional regulation of stress-related responses, whereas cytokinin acts as an anti-senescing agent by maintaining cellular activities and preserving the translational machinery. These findings provide new insights into how plants utilize two antagonistic hormones, ethylene and cytokinin, to regulate the molecular programming of leaf senescence.

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a "NAC troika," govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NAC s, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Brassinosteroids (BRs) are essential phytohormones regulating various developmental and physiological processes during normal growth and development. cog1-3D ( cogwheel1-3D ) was identified as an activation-tagged genetic modifier of bri1-5 , an intermediate BR receptor mutant in Arabidopsis ( Arabidopsis thaliana ). COG1 encodes a Dof-type transcription factor found previously to act as a negative regulator of the phytochrome signaling pathway. cog1-3D single mutants show an elongated hypocotyl phenotype under light conditions. A loss-of-function mutant or inducible expression of a dominant negative form of COG1 in the wild type results in an opposite phenotype. A BR profile assay indicated that BR levels are elevated in cog1-3D seedlings. Quantitative reverse transcription-polymerase chain reaction analyses showed that several key BR biosynthetic genes are significantly up-regulated in cog1-3D compared with those of the wild type. Two basic helix-loop-helix transcription factors, PIF4 and PIF5 , were found to be transcriptionally up-regulated in cog1-3D Genetic analysis indicated that PIF4 and PIF5 were required for COG1 to promote BR biosynthesis and hypocotyl elongation. Chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that COG1 binds to the promoter regions of PIF4 and PIF5 , and PIF4 and PIF5 bind to the promoter regions of key BR biosynthetic genes, such as DWF4 and BR6ox2 , to directly promote their expression. These results demonstrated that COG1 regulates BR biosynthesis via up-regulating the transcription of PIF4 and PIF5 ., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Leaf senescence is not only primarily governed by developmental age but also influenced by various internal and external factors. Although some genes that control leaf senescence have been identified, the detailed regulatory mechanisms underlying integration of diverse senescence-associated signals into the senescence programs remain to be elucidated. To dissect the regulatory pathways involved in leaf senescence, we isolated the not oresara1-1 (nore1-1) mutant showing accelerated leaf senescence phenotypes from an EMS-mutagenized Arabidopsis thaliana population. We found that altered transcriptional programs in defense response-related processes were associated with the accelerated leaf senescence phenotypes observed in nore1-1 through microarray analysis. The nore1-1 mutation activated defense program, leading to enhanced disease resistance. Intriguingly, high ambient temperature effectively suppresses the early senescence and death phenotypes of nore1-1. The gene responsible for the phenotypes of nore1-1 contains a missense mutation in SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1), which was reported as a negative regulator of premature senescence in the light intensity- and PHYTOALEXIN DEFICIENT 4 (PAD4)-dependent manner. Through extensive double mutant analyses, we recently identified suppressor of the G2 Allele of SKP1b (SGT1b), one of the positive regulators for disease resistance conferred by many resistance (R) proteins, as a downstream signaling component in NORE1-mediated senescence and cell death pathways. In conclusion, NORE1/SAUL1 is a key factor integrating signals from temperature-dependent defense programs and leaf senescence in Arabidopsis. These findings provide a new insight that plants might utilize defense response program in regulating leaf senescence process, possibly through recruiting the related genes during the evolution of the leaf senescence program., (© 2016 Scandinavian Plant Physiology Society.)

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

As most organisms age, their appearance, physiology, and behaviour alters as part of a life history strategy that maximizes their fitness over their lifetime. The passage of time is measured by organisms and is used to modulate these age-related changes. Organisms have an endogenous time measurement system called the circadian clock. This endogenous clock regulates many physiological responses throughout the life history of organisms to enhance their fitness. However, little is known about the relation between ageing and the circadian clock in plants. Here, we investigate the association of leaf ageing with circadian rhythm changes to better understand the regulation of life-history strategy in Arabidopsis. The circadian periods of clock output genes were approximately 1h shorter in older leaves than younger leaves. The periods of the core clock genes were also consistently shorter in older leaves, indicating an effect of ageing on regulation of the circadian period. Shortening of the circadian period with leaf age occurred faster in plants grown under a long photoperiod compared with a short photoperiod. We screened for a regulatory gene that links ageing and the circadian clock among multiple clock gene mutants. Only mutants for the clock oscillator TOC1 did not show a shortened circadian period during leaf ageing, suggesting that TOC1 may link age to changes in the circadian clock period. Our findings suggest that age-related information is incorporated into the regulation of the circadian period and that TOC1 is necessary for this integrative process., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant roots anchor the plant to the soil and absorb water and nutrients for growth. Understanding the molecular mechanisms regulating root development is essential for improving plant survival and agricultural productivity. Extensive molecular genetic studies have provided important information on crucial components for the root development control over the last few decades. However, it is becoming difficult to identify new regulatory components in root development due to the functional redundancy and lethality of genes involved in root development. In this study, we performed a chemical genetic screen to identify novel synthetic compounds that regulate root development in Arabidopsis seedlings. The screen yielded a root growth inhibitor designated as 'rootin', which inhibited Arabidopsis root development by modulating cell division and elongation, but did not significantly affect shoot development. Transcript analysis of phytohormone marker genes revealed that rootin preferentially altered the expression of auxin-regulated genes. Furthermore, rootin reduced the accumulation of PIN1, PIN3, and PIN7 proteins, and affected the auxin distribution in roots, which consequently may lead to the observed defects in root development. Our results suggest that rootin could be utilized to unravel the mechanisms underlying root development and to investigate dynamic changes in PIN-mediated auxin distribution., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many of duplicated genes are enriched in signaling pathways. Recently, gene duplication of kinases has been shown to provide genetic buffering and functional diversification in cellular signaling. Transcription factors (TFs) are also often duplicated. However, how duplication of TFs affects their regulatory structures and functions of target genes has not been explored at the systems level. Here, we examined regulatory and functional roles of duplication of three major ARR TFs (ARR1, 10, and 12) in Arabidopsis cytokinin signaling using wild-type and single, double, and triple deletion mutants of the TFs. Comparative analysis of gene expression profiles obtained from Arabidopsis roots in wild-type and these mutants showed that duplication of ARR TFs systematically extended their transcriptional regulatory structures, leading to enhanced robustness and diversification in functions of target genes, as well as in regulation of cellular networks of target genes. Therefore, our results suggest that duplication of TFs contributes to robustness and diversification in functions of target genes by extending transcriptional regulatory structures.

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Phytochromes are red (R)/far-red (FR) photoreceptors that are central to the regulation of plant growth and development. Although it is well known that photoactivated phytochromes are translocated into the nucleus where they interact with a variety of nuclear proteins and ultimately regulate genome-wide transcription, the mechanisms by which these photoreceptors function are not completely understood. In an effort to enhance our understanding of phytochrome-mediated light signaling networks, we attempted to identify novel proteins interacting with phytochrome B (phyB). Using affinity purification in Arabidopsis phyB overexpressor, coupled with mass spectrometry analysis, 16 proteins that interact with phyB in vivo were identified. Interactions between phyB and six putative phyB-interacting proteins were confirmed by bimolecular fluorescence complementation (BiFC) analysis. Involvement of these proteins in phyB-mediated signaling pathways was also revealed by physiological analysis of the mutants defective in each phyB-interacting protein. We further characterized the athb23 mutant impaired in the homeobox protein 23 (ATHB23) gene. The athb23 mutant displayed altered hypocotyl growth under R light, as well as defects in phyB-dependent seed germination and phyB-mediated cotyledon expansion. Taken together, these results suggest that the ATHB23 transcription factor is a novel component of the phyB-mediated R light signaling pathway., (© 2013 Scandinavian Plant Physiology Society.)

Chemical genetics utilizes small molecules to perturb biological processes. Unlike conventional genetics methods, which involve the alteration of genetic information mostly with lasting effects, chemical genetics allows temporary and reversible alterations of biological processes. Furthermore, it enables the alteration of biological processes in a dose-dependent manner, providing an advantage over conventional genetics. In the present chapter, the general procedures of forward chemical genetic screening are described. Forward chemical genetic screening can be performed in three steps. The first step involves the identification of small molecules that induce phenotypic or physiological changes in a biological system from a chemical library. In the second step, cellular targets that interact with the isolated chemical, which are mostly proteins, are identified. Although several methods can be applied in the second step, the most common one is affinity pull-down assay using a target protein that binds to the isolated compound. However, affinity pull-down of a target protein is a formidable barrier in forward chemical genetics. We introduced a tagged chemical library approach that significantly facilitates the identification of target proteins. The third step consists of the validation of the target protein, which should include the assessment of target specificity. This step is critical because small molecules often show pleiotropic effects due to low specificity. The specificity test may include a competition assay using cold competitors and a genetic study using mutants or transgenic lines modified for the cellular target.

Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F v/F m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.

Biological networks consist of a defined set of regulatory motifs. Subcellular compartmentalization of regulatory molecules can provide a further dimension in implementing regulatory motifs. However, spatial regulatory motifs and their roles in biological networks have rarely been explored. Here we show, using experimentation and mathematical modeling, that spatial segregation of GIGANTEA (GI), a critical component of plant circadian systems, into nuclear and cytosolic compartments leads to differential functions as positive and negative regulators of the circadian core gene, LHY, forming an incoherent feedforward loop to regulate LHY. This regulatory motif formed by nucleocytoplasmic partitioning of GI confers, through the balanced operation of the nuclear and cytosolic GI, strong rhythmicity and robustness to external and internal noises to the circadian system. Our results show that spatial and functional segregation of a single molecule species into different cellular compartments provides a means for extending the regulatory capabilities of biological networks., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The endogenous circadian clock regulates many physiological processes related to plant survival and adaptability. GIGANTEA (GI), a clock-associated protein, contributes to the maintenance of circadian period length and amplitude, and also regulates flowering time and hypocotyl growth in response to day length. Similarly, EARLY FLOWERING 4 (ELF4), another clock regulator, also contributes to these processes. However, little is known about either the genetic or molecular interactions between GI and ELF4 in Arabidopsis. In this study, we investigated the genetic interactions between GI and ELF4 in the regulation of circadian clock-controlled outputs. Our mutant analysis shows that GI is epistatic to ELF4 in flowering time determination, while ELF4 is epistatic to GI in hypocotyl growth regulation. Moreover, GI and ELF4 have a synergistic or additive effect on endogenous clock regulation. Gene expression profiling of gi, elf4, and gi elf4 mutants further established that GI and ELF4 have differentially dominant influences on circadian physiological outputs at dusk and dawn, respectively. This phasing of GI and ELF4 influences provides a potential means to achieve diversity in the regulation of circadian physiological outputs, including flowering time and hypocotyl growth.

Leaf senescence, which constitutes the final stage of leaf development, involves programmed cell death and is intricately regulated by various internal and environmental signals that are incorporated with age-related information. ABA plays diverse and important physiological roles in plants, and is involved in various developmental events and stress responses. ABA has long been regarded as a positive regulator of leaf senescence. However, the cellular mediators of ABA-induced senescence have not been identified. We sought to understand the ABA-induced senescence signaling process in Arabidopsis by examining the function of an ABA- and age-induced gene, RPK1, which encodes a membrane-bound, leucine-rich repeat-containing receptor kinase (receptor protein kinase 1). Loss-of-function mutants in RPK1 were significantly delayed in age-dependent senescence. Furthermore, rpk1 mutants exhibited reduced sensitivity to ABA-induced senescence but little change to jasmonic acid- or ethylene-induced senescence. RPK1 thus mediates ABA-induced leaf senescence as well as age-induced leaf senescence. Conditional overexpression of RPK1 at the mature stage clearly accelerated senescence and cell death, whereas induction of RPK1 at an early developmental stage retarded growth without triggering senescence symptoms. Therefore, RPK1 plays different roles at different stages of development. Consistently, exogenously applied ABA affected leaf senescence in old leaves but not in young leaves. The results, together, showed that membrane-bound RPK1 functions in ABA-dependent leaf senescence. Furthermore, the effect of ABA and ABA-inducible RPK1 on leaf senescence is dependent on the age of the plant, which in part explains the mechanism of functional diversification of ABA action.

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.

Phytochrome regulates various physiological and developmental processes throughout the life cycle of plants. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates the far-red light high irradiance response (FR-HIR), which is elicited by continuous far-red light. In FR-HIR, nuclear accumulation of phyA, which precedes physiological responses, is proposed to be required for the response. In contrast to FR, red light induces rapid degradation of phyA to suppress undesirable long-term photomorphogenic responses of phyA. In the present study, we compared biological activities between phyA derivatives to which either a nuclear localization (NLS) or export (NES) signal sequence was attached. Those derivatives were expressed under the control of the PHYA promoter in the Arabidopsis phyA mutant. Detailed microscopic observation revealed that the phyA-green fluorescent protein (GFP) without a signal sequence is localized exclusively in the cytoplasm in darkness. Rapid nuclear entry was observed after exposure to both red and far-red light. Interestingly, both phyA-GFP-NLS and phyA-GFP-NES were rapidly degraded under continuous red light. Furthermore, a proteasome inhibitor delayed degradation equally under these two conditions. Therefore, similar mechanisms for phyA degradation may exist in the cytoplasm and nucleus. As expected from previous reports, phyA-GFP-NLS, but not phyA-GFP-NES, mediated different aspects of FR-HIR, such as inhibition of hypocotyl elongation and rapid induction of gene expression, confirming that phyA nuclear localization is required for FR-HIR. In addition, a detailed time course analysis of phyA-GFP and phyA-GFP-NLS responses revealed that they were almost indistinguishable, raising the question of the physiological relevance of phyA cytoplasmic retention in darkness.

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development and involves the extensive reprogramming of gene expression. Despite the importance of senescence in plants, the underlying regulatory mechanisms are not well understood. This study reports the isolation and functional analysis of RAV1, which encodes a RAV family transcription factor. Expression of RAV1 and its homologues is closely associated with leaf maturation and senescence. RAV1 mRNA increased at a later stage of leaf maturation and reached a maximal level early in senescence, but decreased again during late senescence. This profile indicates that RAV1 could play an important regulatory role in the early events of leaf senescence. Furthermore, constitutive and inducible overexpression of RAV1 caused premature leaf senescence. These data strongly suggest that RAV1 is sufficient to cause leaf senescence and it functions as a positive regulator in this process.

Auxin regulates a variety of physiological and developmental processes in plants. Although auxin acts as a suppressor of leaf senescence, its exact role in this respect has not been clearly defined, aside from circumstantial evidence. It was found here that ARF2 functions in the auxin-mediated control of Arabidopsis leaf longevity, as discovered by screening EMS mutant pools for a delayed leaf senescence phenotype. Two allelic mutations, ore14-1 and 14-2, caused a highly significant delay in all senescence parameters examined, including chlorophyll content, the photochemical efficiency of photosystem II, membrane ion leakage, and the expression of senescence-associated genes. A delay of senescence symptoms was also observed under various senescence-accelerating conditions, where detached leaves were treated with darkness, phytohormones, or oxidative stress. These results indicate that the gene defined by these mutations might be a key regulatory genetic component controlling functional leaf senescence. Map-based cloning of ORE14 revealed that it encodes ARF2, a member of the auxin response factor (ARF) protein family, which modulates early auxin-induced gene expression in plants. The ore14/arf2 mutation also conferred an increased sensitivity to exogenous auxin in hypocotyl growth inhibition, thereby demonstrating that ARF2 is a repressor of auxin signalling. Therefore, the ore14/arf2 lesion appears to cause reduced repression of auxin signalling with increased auxin sensitivity, leading to delayed senescence. Altogether, our data suggest that ARF2 positively regulates leaf senescence in Arabidopsis.

Cytosolic Ca(2+) ([Ca(2+)](cyt)) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca(2+)](cyt) has been shown to regulate stomatal movements, and a defined window of Ca(2+) oscillation kinetic parameters encodes necessary information for long-term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca(2+) signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over-expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca(2+)-induced stomatal closure. Cytosolic Ca(2+) activation of S-type anion channels, which play a central role in Ca(2+)-reactive stomatal closure, was normal in the AtGLR3.1 over-expressing plants. Interestingly, AtGLR3.1 over-expression did not affect Ca(2+)-induced Ca(2+) oscillation kinetics, but resulted in a failure to maintain long-term 'Ca(2+)-programmed' stomatal closure when Ca(2+) oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short-term Ca(2+)-reactive closure was not affected in AtGLR3.1 over-expressing plants. In wild-type plants, the translational inhibitor cyclohexamide partially inhibited Ca(2+)-programmed stomatal closure induced by experimentally imposed Ca(2+) oscillations without affecting short-term Ca(2+)-reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over-expressing plants. Our results suggest that over-expression of AtGLR3.1 impairs Ca(2+) oscillation-regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long-term Ca(2+)-programmed stomatal closure.

Aging induces gradual yet massive cell death in higher organisms, including annual plants. Even so, the underlying regulatory mechanisms are barely known, despite the long-standing interest in this topic. Here, we demonstrate that ORE1, which is a NAC (NAM, ATAF, and CUC) transcription factor, positively regulates aging-induced cell death in Arabidopsis leaves. ORE1 expression is up-regulated concurrently with leaf aging by EIN2 but is negatively regulated by miR164. miR164 expression gradually decreases with aging through negative regulation by EIN2, which leads to the elaborate up-regulation of ORE1 expression. However, EIN2 still contributes to aging-induced cell death in the absence of ORE1. The trifurcate feed-forward pathway involving ORE1, miR164, and EIN2 provides a highly robust regulation to ensure that aging induces cell death in Arabidopsis leaves.

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex (SCF(FBL17)) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCF(FBL17) as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.

Cryptochromes (CRY) are one of the two major classes of photoreceptors that perceive light stimuli in the UV-A to blue light region and they are involved in multiple aspects of plant growth and development. However, knowledge regarding their signaling transduction components and mechanisms remains limited. Here, we report that a MYB transcription factor Blue Insensitive Trait 1 (BIT1), plays an important role in controlling blue light responses. Hypocotyl growth responses indicate that BIT1 functions as a positive element in blue light signaling, since BIT1 antisense and knock-out lines show a reduced light response in blue light. BIT1 controls blue light-dependent expression of various genes such as PsbS, a member of the light-harvesting complex gene family. A transactivation assay showed that BIT1 regulates promoter activity of PsbS in a blue light-dependent manner and that it requires CRY1 for activation of the PsbS promoter. BIT1 undergoes degradation in darkness and CRY1 functions to stabilize BIT1 in a blue light-dependent manner. In contrast, COP1 binds to BIT1 and mediates its degradation. We propose that the PsbS promoter is activated in blue light via the blue light-dependent stabilization of BIT1 by CRY1, while in darkness BIT1 is degraded by COP1-mediated proteolysis.

In plants, the circadian clock controls daily physiological cycles as well as daylength-dependent developmental processes such as photoperiodic flowering and seedling growth. Here, we report that FIONA1 (FIO1) is a genetic regulator of period length in the Arabidopsis thaliana circadian clock. FIO1 was identified by screening for a mutation in daylength-dependent flowering. The mutation designated fio1-1 also affects daylength-dependent seedling growth. fio1-1 causes lengthening of the free-running circadian period of leaf movement and the transcription of various genes, including the central oscillators CIRCADIAN CLOCK-ASSOCIATED1, LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, and LUX ARRHYTHMO. However, period lengthening is not dependent upon environmental light or temperature conditions, which suggests that FIO1 is not a simple input component of the circadian system. Interestingly, fio1-1 exerts a clear effect on the period length of circadian rhythm but has little effect on its amplitude and robustness. FIO1 encodes a novel nuclear protein that is highly conserved throughout the kingdoms. We propose that FIO1 regulates period length in the Arabidopsis circadian clock in a close association with the central oscillator and that the circadian period can be controlled separately from amplitude and robustness.

Leaf senescence is the final stage of leaf development and is finely regulated via a complex genetic regulatory network incorporating both developmental and environmental factors. In an effort to identify negative regulators of leaf senescence, we screened activation-tagged Arabidopsis lines for mutants that exhibit a delayed leaf senescence phenotype. One of the mutants (ore7-1D) showed a highly significant delay of leaf senescence in the heterozygous state, leading to at least a twofold increase in leaf longevity. The activated gene (ORE7/ESC) encoded a protein with an AT-hook DNA-binding motif; such proteins are known to co-regulate transcription of genes through modification of chromatin architecture. We showed that ORE7/ESC, in addition to binding to a plant AT-rich DNA fragment, could also modify the chromatin architecture, as illustrated by an altered distribution of a histone-GFP fusion protein in the nucleus of the mutant. Globally altered gene expression, shown by microarray analysis, also indicated that activation of ORE7/ESC results in a younger condition in the mutant leaves. We propose that ectopically expressed ORE7/ESC is negatively regulating leaf senescence and suggest that the resulting chromatin alteration may have a role in controlling leaf longevity. Interestingly, activation of ORE7/ESC also led to a highly extended post-harvest storage life.

The circadian clock is essential for coordinating the proper phasing of many important cellular processes. Robust cycling of key clock elements is required to maintain strong circadian oscillations of these clock-controlled outputs. Rhythmic expression of the Arabidopsis thaliana F-box protein ZEITLUPE (ZTL) is necessary to sustain a normal circadian period by controlling the proteasome-dependent degradation of a central clock protein, TIMING OF CAB EXPRESSION 1 (TOC1). ZTL messenger RNA is constitutively expressed, but ZTL protein levels oscillate with a threefold change in amplitude through an unknown mechanism. Here we show that GIGANTEA (GI) is essential to establish and sustain oscillations of ZTL by a direct protein-protein interaction. GI, a large plant-specific protein with a previously undefined molecular role, stabilizes ZTL in vivo. Furthermore, the ZTL-GI interaction is strongly and specifically enhanced by blue light, through the amino-terminal flavin-binding LIGHT, OXYGEN OR VOLTAGE (LOV) domain of ZTL. Mutations within this domain greatly diminish ZTL-GI interactions, leading to strongly reduced ZTL levels. Notably, a C82A mutation in the LOV domain, implicated in the flavin-dependent photochemistry, eliminates blue-light-enhanced binding of GI to ZTL. These data establish ZTL as a blue-light photoreceptor, which facilitates its own stability through a blue-light-enhanced GI interaction. Because the regulation of GI transcription is clock-controlled, consequent GI protein cycling confers a post-translational rhythm on ZTL protein. This mechanism of establishing and sustaining robust oscillations of ZTL results in the high-amplitude TOC1 rhythms necessary for proper clock function.

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.

Light critically affects the physiology of plants. Using two-dimensional gel electrophoresis, we used a proteomics approach to analyze the responses of Arabidopsis thaliana to red (660 nm), far-red (730 nm) and blue (450 nm) light, which are utilized by type II and type I phytochromes, and blue light receptors, respectively. Under specific light treatments, the proteomic profiles of 49 protein spots exhibited over 1.8-fold difference in protein abundance, significant at p <0.05. Most of these proteins were metabolic enzymes, indicating metabolic changes induced by light of specific wavelengths. The differentially-expressed proteins formed seven clusters, reflecting co-regulation. We used the 49 differentially-regulated proteins as molecular markers for plant responses to light, and by developing a procedure that calculates the Pearson correlation distance of cluster-to-cluster similarity in expression changes, we assessed the proteome-based relatedness of light responses for wild-type and phytochrome mutant plants. Overall, this assessment was consistent with the known physiological responses of plants to light. However, we also observed a number of novel responses at the proteomic level, which were not predicted from known physiological changes.

Cytokinins are plant hormones with profound roles in growth and development, including control of leaf longevity. Although the cytokinin signal is known to be perceived by histidine kinase receptors, the underlying molecular mechanism and specificity of the receptors leading to delayed leaf senescence have not yet been elucidated. Here, we found that AHK3, one of the three cytokinin receptors in Arabidopsis, plays a major role in controlling cytokinin-mediated leaf longevity through a specific phosphorylation of a response regulator, ARR2. This result was obtained through identification of a gain-of-function Arabidopsis mutant that shows delayed leaf senescence because of a missense mutation in the extracellular domain of AHK3. A loss-of-function mutation in AHK3, but not of the other cytokinin receptors, conferred a reduced sensitivity to cytokinin in cytokinin-dependent delay of leaf senescence and abolished cytokinin-dependent phosphorylation of ARR2. Consistently, transgenic overexpression of wild-type, but not an unphosphorylatable mutant ARR2, led to delayed senescence of leaves.

An analysis of changes in global gene expression patterns during developmental leaf senescence in Arabidopsis has identified more than 800 genes that show a reproducible increase in transcript abundance. This extensive change illustrates the dramatic alterations in cell metabolism that underpin the developmental transition from a photosynthetically active leaf to a senescing organ which functions as a source of mobilizable nutrients. Comparison of changes in gene expression patterns during natural leaf senescence with those identified, when senescence is artificially induced in leaves induced to senesce by darkness or during sucrose starvation-induced senescence in cell suspension cultures, has shown not only similarities but also considerable differences. The data suggest that alternative pathways for essential metabolic processes such as nitrogen mobilization are used in different senescent systems. Gene expression patterns in the senescent cell suspension cultures are more similar to those for dark-induced senescence and this may be a consequence of sugar starvation in both tissues. Gene expression analysis in senescing leaves of plant lines defective in signalling pathways involving salicylic acid (SA), jasmonic acid (JA) and ethylene has shown that these three pathways are all required for expression of many genes during developmental senescence. The JA/ethylene pathways also appear to operate in regulating gene expression in dark-induced and cell suspension senescence whereas the SA pathway is not involved. The importance of the SA pathway in the senescence process is illustrated by the discovery that developmental leaf senescence, but not dark-induced senescence, is delayed in plants defective in the SA pathway.

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

The life of a leaf initiated from a leaf primordium ends with senescence, the final step of leaf development. Leaf senescence is a developmentally programmed degeneration process that is controlled by multiple developmental and environmental signals. It is a highly regulated and complex process that involves orderly, sequential changes in cellular physiology, biochemistry, and gene expression. Elucidating molecular mechanisms underlying such a complex, yet delicate process of leaf senescence is a challenging and important biological task. For the past decade, impressive progress has been achieved on the molecular processes of leaf senescence through identification of genes that show enhanced expression during senescence. In addition, Arabidopsis has been established as a model plant for genetic analysis of leaf senescence. The progress on the characterization of genetic mutants of leaf senescence in Arabidopsis has firmly shown that leaf senescence is a genetically controlled developmental phenomenon involving numerous regulatory elements. Especially, employment of global expression analysis as well as genomic resources in Arabidopsis has been very fruitful in revealing the molecular genetic nature and mechanisms underlying leaf senescence. This progress, including molecular characterization of some of the genetic regulatory elements, are revealing that senescence is composed of a complex regulatory network. In this review, we will present current understanding of the molecular genetic mechanisms by which leaf senescence is regulated and processed, focusing mostly on the regulatory factors of senescence in Arabidopsis. We also present a potential biotechnological implication of leaf senescence studies on the improvement of important agronomic traits such as crop yield and post-harvest shelf life. We further provide future research prospects to better understand the complex regulatory network of senescence.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.

Reactive oxygen species play a critical role in mediating the oxidative damage that causes senescence in a variety of aerobic organisms, from yeast to mammals. Genetic studies of these organisms have revealed that extended longevity is frequently associated with an increased resistance to stress. However, the relationship between life span and oxidative stress tolerance in plants is poorly understood. We have investigated the responses to oxidative stress in the delayed leaf senescence mutants of Arabidopsis thaliana, ore1, ore3, and ore9. The detached leaves of these mutants exhibit increased tolerance to various types of oxidative stress. The ore1, ore3, and ore9 mutants were also more tolerant to oxidative stress at the level of the whole plant, as determined by measuring physiological and molecular changes associated with oxidative stress. However, the activities of antioxidant enzymes were similar or lower in the mutants, as compared to wild type. These results suggest that the increased resistance to oxidative stress in the ore1, ore3, and ore9 mutants is not due to enhanced activities of these antioxidant enzymes. Taken together, our findings provide genetic evidence that oxidative stress tolerance is linked to control of leaf longevity in plants.

All organisms, including plants, perceive environmental stress, and they use this information to modify their behavior or development. Here, we demonstrate that Arabidopsis plants have memory functions related to repeated exposure to stressful concentrations of the phytohormone abscisic acid (ABA), which acts as a chemical signal. Repeated exposure of plants to ABA (40 micro m for 2 h) impaired light-induced stomatal opening or inhibited the response to a light stimulus after ABA-entrainment under both dark/light cycle and continuous light. Moreover, there were transient expressions of the rd22 gene during the same periods under both the growing conditions. Such acquired information in ABA-entrained plants produced a long-term sensitization. When the time of light application was changed, a transient induction of the rd22 gene in plants after ABA-entrainment indicated that these were light-associated responses. These transient effects were also observed in kin1, rab18, and rd29B. The transient expression of AtNCED3, causing the accumulation of endogenous ABA, indicated a possible regulation by ABA-dependent pathways in ABA-entrained plants. An ABA immunoassay supported this hypothesis: ABA-entrained plants showed a transient increase in endogenous ABA level from 220 to 250 pmol g-1 fresh mass at 1-2 h of the training period, whereas ABA-deficient (aba2) mutants did not. Taking into account these results, we propose that plants have the ability to memorize stressful environmental experiences, and discuss the molecular events in ABA-entrained plants.

Leaf senescence is a developmentally programmed degeneration process that constitutes the final step of leaf development and is controlled by multiple developmental and environmental signals. In addition to the information obtained from other plants, Arabidopsis has, as a model system, contributed to our understanding of this complex phenomenon in molecular genetic terms. Recent discoveries have identified several genetic mutants and potential regulatory components in Arabidopsis. Identifying further mutants that exploit novel biological resources, screening Scheme and a global functional analysis of senescence-associated genes in Arabidopsis should increase our understanding of the complex regulatory networks.

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.

Light is a critical environmental factor that influences almost all developmental aspects of plants, including seed germination, seedling morphogenesis, and transition to reproductive growth. Plants have therefore developed an intricate network of mechanisms to perceive and process environmental light information. To further characterize the molecular basis of light-signaling processes in plants, we screened an activation tagging pool of Arabidopsis for altered photoresponses. A dominant mutation, cog1-D, attenuated various red (R) and far-red (FR) light-dependent photoresponses. The mutation was caused by overexpression of a gene encoding a member of the Dof family of transcription factors. The photoresponses in Arabidopsis were inversely correlated with the expression levels of COG1 mRNA. When the COG1 gene was overexpressed in transgenic plants, the plants exhibited hyposensitive responses to R and FR light in a manner inversely dependent on COG1 mRNA levels. On the other hand, transgenic lines expressing antisense COG1 were hypersensitive to R and FR light. Expression of the COG1 gene is light inducible and requires phytochrome A (phyA) for FR light-induced expression and phytochrome B (phyB) for R light-induced expression. Thus, the COG1 gene functions as a negative regulator in both the phyA- and phyB-signaling pathways. We suggest that these phytochromes positively regulate the expression of COG1, a negative regulator, as a mechanism for fine tuning the light-signaling pathway.