Biomedical subjects

Hyun-Sook Pai

Publications and source records attributed to Hyun-Sook Pai.

23 records · Page 2Linked to original sources

PubMed2003

Activation of the programmed cell death pathway by inhibition of proteasome function in plants.

Proteasomes constitute the major machinery to degrade or process proteins by ATP/ubiquitin-mediated proteolysis. Recent findings suggest a pivotal role of the ubiquitin/proteasome pathway in the regulation of apoptosis in animal cells. Here we show that virus-induced gene silencing of two different subunits of the 26 S proteasome, the alpha 6 subunit of the 20 S proteasome and RPN9 subunit of 19 S regulatory complex, both activated the programmed cell death (PCD) program, accompanied by reduced proteasome activity and accumulation of polyubiquitinated proteins. These results demonstrate that disruption of proteasome function leads to PCD in plant cells. The affected cells showed morphological markers of PCD, including nuclear condensation and DNA fragmentation, accompanied by the 10-fold higher production of reactive oxygen species and increased ion leakage for 3-fold. Similar to apoptosis in animal system, mitochondrial membrane potential was decreased, cytochrome c released from mitochondria to cytosol, and caspase 9- and caspase 3-like proteolytic activities detected in the cells. Interestingly, this proteasome-mediated PCD stimulated the expression of only a subset of transcripts that are highly induced during pathogen-mediated hypersensitive response cell death, indicating that the two PCD pathways are differentially regulated. Taken together, these results provide the first direct evidence that proteasomes play a role in the regulatory program of PCD in plants. Controlled inhibition of proteasome activities may be involved in developmentally or environmentally activated plant cell death programs.

Apoptosis↗

PubMed2003

Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana.

The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.

Abscisic Acid↗

PubMed2003

CHRK1, a chitinase-related receptor-like kinase, plays a role in plant development and cytokinin homeostasis in tobacco.

CHRK1 encodes a receptor-like kinase that contains a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. In this study, we showed that CHRK1 is mainly expressed in the shoot apex region including leaf primordia and young leaves, and germinating seedlings and vascular tissues, based on GUS activity of transgenic tobacco plants carrying the CHRK1 promoter-GUS fusion gene. Transgenic tobacco plants in which CHRK1 expression was suppressed exhibited pleiotrophic developmental abnormality, including formation of proliferating shooty calli from emerging seedlings and severely altered seedling development. At the cellular level, ectopic cell proliferation, reduced cell specificity, and aberrant chloroplast development were observed. The transgenic lines contained 3-fold higher level of cytokinin than the wild-type plants. Consistently, the transgenic seedlings exhibited a typical cytokinin response in the absence of hormone, such as deetiolation under the dark. Based on these results, we propose that CHRK1 is involved in a developmental signaling pathway regulating cell proliferation/differentiation and the endogenous cytokinin levels in tobacco.

Cell Division↗

PubMed2003

Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum.

Using a yeast two-hybrid system, we identified NtRpn3, a regulatory subunit of 26S proteasome, as an interacting protein of NtCDPK1 calcium-dependent protein kinase in Nicotiana tabacum. Rpn3 in yeast is an essential protein involved in proteolysis of cell cycle regulatory proteins, and the carrot homolog of Rpn3 was previously isolated as a nuclear antigen that is mainly expressed in the meristem. NtCDPK1 physically interacts with NtRpn3 in vitro in a Ca2+-independent manner and phosphorylates NtRpn3 in a Ca2+-dependent manner with Mg2+ as a cofactor. NtCDPK1 and NtRpn3 are co-localized in the nucleus, nuclear periphery, and around plasma membrane in vivo. Both NtCDPK1 and AtRpn3, an NtRpn3 homolog of Arabidopsis, are mainly expressed in the rapidly proliferating tissues including shoot and root meristems, and developing floral buds. Virus-induced gene silencing of either NtRpn3 or NtCDPK1 resulted in the phenotypes of abnormal cell morphology and premature cell death in newly emerged leaves. Finally, NtCDPK1 interacts with NtRpn3 in vivo as shown by co-immunoprecipitation. Based on these results, we propose that NtCDPK1 and NtRpn3 are interacting in a common signal transduction pathway possibly for regulation of cell division, differentiation, and cell death in tobacco.

Calcium↗

PubMed2002

Forkhead-associated domains of the tobacco NtFHA1 transcription activator and the yeast Fhl1 forkhead transcription factor are functionally conserved.

NtFHA1 encodes a novel protein containing the forkhead-associated (FHA) domain and the acidic domain in Nicotiana tabacum. NtFHA1 functions as a transactivator and is targeted to the nucleus. The sequence of the FHA domain of NtFHA1 is significantly homologous to that of the Fhl1 forkhead transcription factor of yeast. FHL1 was previously identified as a suppressor of RNA polymerase III mutations, and the fhl1 deletion mutant exhibited severe growth defects and impaired rRNA processing. Ectopic expression of the FHA domain of NtFHA1 (but not its mutant form) resulted in severe growth retardation in yeast. Similarly, expression of Fhl1, its FHA domain, or chimeric Fhl1 containing the NtFHA1 FHA domain also inhibited yeast growth. Yeast cells overexpressing the FHA domains of NtFHA1 and Fhl1 contained lower levels of mature rRNAs and exhibited rRNA-processing defects, similar to the fhl1 null mutant. Chimeric Fhl1 (but not the mutant form with a small deletion in its FHA domain) fully complemented the growth and rRNA-processing defects of the fhl1 null mutant, demonstrating that the FHA domain of NtFHA1 can functionally substitute for the FHA domain of Fhl1. These results demonstrate that the FHA domains of NtFHA1 and Fhl1 are conserved in their structure and function and that the FHA domain of Fhl1 is critically involved in regulation of rRNA processing in yeast. NtFHA1 function in plants may be analogous to Fhl1 function in yeast.

Amino Acid Sequence↗