Hong Gil Nam

Age-Dependent Action of an ABA-Inducible Receptor Kinase, RPK1, as a Positive Regulator of Senescence in Arabidopsis Leaves

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.

ORE9, an F-Box Protein That Regulates Leaf Senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693–amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.

Trifurcate Feed-Forward Regulation of Age-Dependent Cell Death Involving miR164 in Arabidopsis

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.

Molecular genetics of leaf senescence in Arabidopsis

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.

The molecular genetic analysis of leaf senescence

The cloning of genes induced during leaf senescence and the study of their modes of regulation conducted in the past two years have revealed some of the molecular mechanisms underlying leaf senescence. The identification of genetic mutants that control leaf senescence in Arabidopsis thaliana opened up new possibilities for genetically analyzing leaf senescence in a model system. Encouraging experimental data with transgenic plants show that manipulation of leaf senescence may greatly contribute to the improvement of important agronomic traits such as crop yield and the storage life of the harvested tissues.

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5′-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency.

Identification of a Receptor-Like Protein Kinase Gene Rapidly Induced by Abscisic Acid, Dehydration, High Salt, and Cold Treatments in Arabidopsis thaliana

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1–1, abi2–1, and abi3–1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed.

Restriction Fragment Length Polymorphism Linkage Map of Arabidopsis thaliana.

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.

Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in Arabidopsis

Four cDNA clones, named pSEN2, pSEN3, pSEN4, and pSEN5, for mRNAs induced during leaf senescence in Arabidopsis thaliana were characterized. The clones were isolated from a cDNA library of detached leaves incubated in darkness for 2 days to accelerate senescence, first by differential screening and then by examining expression of the primarily screened clones during age-dependent leaf senescence. Transcript levels detected by these cDNA clones, thus, were up-regulated in an age-dependent manner and during dark-induced leaf senescence. In contrast, when leaf senescence was induced by ethylene, ABA or methyl jasmonate, the transcript level detected by the clones was differentially regulated depending on the senescence-inducing hormones. The transcript level for pSEN4 increased during senescence induced by all three hormones, while the transcript detected by the pSEN2 clone did not increase during senescence induced by ethylene. The transcript level for pSEN5 was increased upon ABA-induced senescence but decreased during ethylene-induced senescence. The pSEN3 clone detected multiple transcripts that are differentially regulated by these factors. The results show that, although the apparent senescence symptoms of Arabidopsis leaf appear similar regardless of the senescence-inducing factors, the detailed molecular state of leaf cells during senescence induced by different senescence-inducing factors is different. The pSEN3 clone encodes a polyubiquitin and the pSEN4 clone encodes a peptide related to endoxyloglucan transferase. This result is consistent with the expected roles of senescence-induced genes during leaf senescence.

OASIS: online application for the survival analysis of lifespan assays performed in aging research.

Background Aging is a fundamental biological process. Characterization of genetic and environmental factors that influence lifespan is a crucial step toward understanding the mechanisms of aging at the organism level. To capture the different effects of genetic and environmental factors on lifespan, appropriate statistical analyses are needed. Methodology/Principal Findings We developed an online application for survival analysis (OASIS) that helps conduct various novel statistical tasks involved in analyzing survival data in a user-friendly manner. OASIS provides standard survival analysis results including Kaplan-Meier estimates and mean/median survival time by taking censored survival data. OASIS also provides various statistical tests including comparison of mean survival time, overall survival curve, and survival rate at specific time point. To visualize survival data, OASIS generates survival and log cumulative hazard plots that enable researchers to easily interpret their experimental results. Furthermore, we provide statistical methods that can analyze variances among survival datasets. In addition, users can analyze proportional effects of risk factors on survival. Conclusions/Significance OASIS provides a platform that is essential to facilitate efficient statistical analyses of survival data in the field of aging research. Web application and a detailed description of algorithms are accessible from http://sbi.postech.ac.kr/oasis.