Suk Whan Hong

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.

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.

A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress.

In response to environmental challenges, plant cells activate several signaling pathways that trigger the expression of transcription factors. Arabidopsis MYB60 was reported to be involved in stomatal regulation under drought conditions. Here, two splice variants of the MYB60 gene are shown to play a crucial role in stomatal movement. This role was demonstrated by over-expressing each variant, resulting in enhanced sensitivity to water deficit stress. The MYB60 splice variants, despite the fact that one of which lacks the first two exons encoding the first MYB DNA binding domain, both localize to the nucleus and promote guard cell deflation in response to water deficit. Moreover, MYB60 expression is increased in response to a low level of ABA and decreased in response to high level of ABA. At initial stage of drought stress, the plant system may modulate the root growth behavior by regulating MYB60 expression, thus promotes root growth for increased water uptake. In contrast, severe drought stress inhibits the expression of the MYB60 gene, resulting in stomatal closure and root growth inhibition. Taken together, these data indicate that MYB60 plays a dual role in abiotic stress responses in Arabidopsis through its involvement in stomatal regulation and root growth.

Evaluation of 515 expressed sequence tags obtained from guard cells of Brassica campestris

Abstract. As an attempt to examine the transcripts expressed in a single cell type and to unveil the physiology of guard cells at the molecular level, we generated 515 expressed sequence tags (ESTs) from a directional cDNA library constructed from guard-cell protoplasts of Brassica campestris L. ssp. pekinensis. A comparative analysis of the guard-cell ESTs against the National Center for Biotechnological Information non-redundant protein database revealed that 133 ESTs (26%) have significant similarity to protein coding sequences in the database. Among them were 35 clones related to genes that have not yet been identified in higher plants. Analysis of RNA gel blots of 14 database-matched clones revealed that five clones harbor the sequences for mRNAs expressed most abundantly in guard cells, one of them detecting an mRNA with highly preferential expression in guard cells. Functional categorization of the putatively identified guard-cell ESTs showed, when compared with maize leaf ESTs, that guard cells expressed a higher proportion of signal transduction components and a lower proportion of structural or photosynthetic genes, as is consistent with the roles of guard cells.

Loss of the R2R3 MYB, AtMyb73, causes hyper-induction of the SOS1 and SOS3 genes in response to high salinity in Arabidopsis

Environmental stressors, including high salt, drought, and low or high temperatures, are often associated with significant losses in agricultural productivity. Plants have evolved a diverse array of signaling pathways to modulate their development in response to various environmental challenges. Here, we report the characterization of a member of the R2R3-MYB transcription factor family, AtMyb73. The expression of AtMyb73 was up-regulated by salt stress but not by other stresses. The maximum level of AtMyb73 expression occurred at 6h of 300mM NaCl treatment. Under salt stress, atmyb73 ko mutant plants exhibited higher survival rates compare to wild type (Col-0) plants. Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, we determined that the accumulation of salt overly sensitive (SOS) transcripts, SOS1 and SOS3, was higher in atmyb73 ko and atmyb73 eko plants than in wild type plants in response to 300mM NaCl treatment. These results indicate that AtMyb73 is a negative regulator of SOS induction in response to salt stress in Arabidopsis thaliana.

Ectopic expression of Expansin3 or Expansinβ1 causes enhanced hormone and salt stress sensitivity in Arabidopsis

Expansins are cell wall loosening proteins that appear to permit the microfibril matrix network to slide in growing plant cell walls, thereby enabling the wall to expand. To scrutinize possible impacts on plant growth and development when expansins are over-expressed, we characterized phenotypic alterations of the transgenic plants that constitutively expressed AtEXP3 or AtEXP-β1 under control of 35S-CaMV promoter. Our results suggest that both AtEXP3-OX and AtXPβ1-OX are very sensitive to salt stress. However, the mechanisms underlying their enhanced salt sensitivity appear to be different.

Two putative protein kinases from Arabidopsis thaliana contain highly acidic domains.

Two cDNA clones (ASK1 and ASK2) for plant protein kinases were cloned from Arabidopsis thaliana by screening cDNA libraries with a degenerate oligonucleotide probe that corresponds to a highly conserved motif among protein kinases. Sequence analysis shows that the clones contain open reading frames that encode 41.2 kDa (ASK1) and 40.1 kDa (ASK2) proteins, respectively. These coding regions contain all the conserved motifs of protein kinases. Structural analysis of the coding regions revealed that the two protein kinase genes share high sequence similarity to each other (76.6% identity). The catalytic domain located in the amino terminal region is most similar to the calcium/calmodulin-dependent protein kinase subfamily (47.2% to 54.2% similarity) and the SNF1 kinase subfamily (48.1% to 53.3% similarity). However, the carboxy terminal regions contain distinctive stretches of 21 (ASK1) and 19 (ASK2) acidic amino acids. These clones are the first report of protein kinases with such acidic amino acid regions. The transcripts of both genes are most abundant in leaf but are also expressed in other organs. The expression of the two genes is highly affected by light regime.

MicroRNA400-guided Cleavage of Pentatricopeptide Repeat Protein mRNAs Renders Arabidopsis thaliana More Susceptible to Pathogenic Bacteria and Fungi

Although a large number of microRNAs (miRNAs) have been identified in different plant species, the functional roles and targets of the majority of miRNAs have not yet been determined. Here, Arabidopsis thaliana miRNA400 (miR400) was investigated for its functional role in the defense response to diverse pathogens. Transgenic Arabidopsis plants that overexpress MIR400 (35S::MIR400) displayed much more severe disease symptoms than the wild-type plants when infected with the bacterium Pseudomonas syringae pv. tomato DC3000 or the fungus Botrytis cinerea. MiR400 guided the cleavage of two genes (At1g06580 and At1g62720) encoding pentatricopeptide repeat (PPR) proteins. To confirm further that the miR400-mediated defense response was due to the cleavage of PPR mRNAs, loss-of-function mutant and artificial miRNA-mediated knockdown mutants of PPR were generated, and their disease responses were analyzed upon pathogen challenge. Similar to the 35S::MIR400 plants, the ppr mutants displayed much more severe disease symptoms than the wild-type plants when challenged with the pathogens, indicating that miR400 affects the defense response by cleaving PPR mRNAs. Expression of miR400 was down-regulated, whereas the PPR1 and PPR2 transcripts increased upon pathogen challenge. Collectively, the present study reveals that miR400-mediated dysfunction of PPR proteins renders Arabidopsis more susceptible to pathogenic bacteria and fungi, which emphasizes the importance of PPR proteins in plant defense against diverse pathogens.

Functional complementation of a yeast vesicular transport mutation ypt1-1 by a Brassica napus cDNA clone encoding a small GTP-binding protein

A cDNA clone (bra) encoding a small GTP-binding protein was isolated from Brassica napus by screening a root cDNA library with a degenerate oligonucleotide probe that corresponds to a highly conserved GTP-binding domain of the Ras superfamily. Sequence analysis shows that the clone contains an open reading frame of 219 amino acid residues with the estimated molecular mass of 24379 Da and this coding region contains all the conserved motifs of the Ras superfamily. The deduced amino acid sequence of the bra gene is most closely related to the Ypt/Rab family that functions in the vesicular transport (46% and 47% amino acid identity to the yeast Ypt1 and to the human Rab1, respectively) and is more distantly related to the other Ras-related families. The protein encoded by the bra gene, when expressed in Escherichia coli, shows the ability to bind GTP. Furthermore, when the bra gene is introduced into Saccharomyces cerevisiae under the regulation of the yeast GAL1 promoter, the gene can complement the temperature-sensitive yeast mutation ypt1-1 that has defects in vesicular transport function. The amino acid sequence similarity and the functional complementation of the yeast mutation suggest that this gene is likely to be involved in the vesicular transport in plants. Genomic Southern analysis shows that this gene is a member of a small gene family in Brassica napus.

The ethylene signaling pathway has a negative impact on sucrose-induced anthocyanin accumulation in Arabidopsis

In an attempt to understand the complex regulatory mechanisms underlying sucrose-induced flavonoid biosynthesis, we examined several Arabidopsis mutants with altered anthocyanin accumulation. We determined that disruption of ethylene signaling results in a dramatic increase in sucrose-induced anthocyanin accumulation. Furthermore, we investigated why the ein2-1 (ethylene insensitive) Arabidopsis mutant accumulates higher levels of anthocyanin in response to sucrose than wild-type Arabidopsis. An increased level of PAP1 transcript in the ein2-1 mutant appears to be the main factor responsible for the increased accumulation of anthocyanin in response to sucrose. Therefore, our results indicate that the ethylene signaling pathway plays a negative role in sucrose-induced anthocyanin accumulation. We believe that the explanation for this observation may be related to the initiation of the senescence program in plants.