Mi Ri Kim

Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis

Environmental challenges to plants typically entail retardation of vegetative growth and delay or cessation of flowering. Here we report a link between the flowering time regulator, GIGANTEA (GI), and adaptation to salt stress that is mechanistically based on GI degradation under saline conditions, thus retarding flowering. GI, a switch in photoperiodicity and circadian clock control, and the SNF1-related protein kinase SOS2 functionally interact. In the absence of stress, the GI:SOS2 complex prevents SOS2-based activation of SOS1, the major plant Na(+)/H(+)-antiporter mediating adaptation to salinity. GI overexpressing, rapidly flowering, plants show enhanced salt sensitivity, whereas gi mutants exhibit enhanced salt tolerance and delayed flowering. Salt-induced degradation of GI confers salt tolerance by the release of the SOS2 kinase. The GI-SOS2 interaction introduces a higher order regulatory circuit that can explain in molecular terms, the long observed connection between floral transition and adaptive environmental stress tolerance in Arabidopsis.

A novel thiol-reductase activity of Arabidopsis YUC6 confers drought tolerance independently of auxin biosynthesis

YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses.

NADPH-dependent thioredoxin reductase A (NTRA) confers elevated tolerance to oxidative stress and drought.

NADPH-dependent thioredoxin reductases (NTRs) are key-regulatory enzymes determining the redox state of the thioredoxin (Trx) system that provides reducing power to peroxidases or oxidoreductases. Moreover, it also plays an essential function in the direct reduction of ROS and acquiring stress tolerance in plant. Cytoplasmic NTRA, mitochondrial NTRB, and chloroplastic NTRC are the three conserved NTRs which cooperate with specific sub-cellularly localized Trxs in Arabidopsis. However, cytosolic NTRs such as NTRA in Arabidopsis have not previously been identified in plants or mammals as a source of functional redundancy with mitochondrial NTRs. Here, we show the involvement of NTRA in the plant stress response counteracting oxidative and drought stresses. Methyl viologen (MV), an inducer of oxidative stress in plants, enhanced the NTRA transcripts. To identify the physiological role of NTRA influencing ROS homeostasis by stress, NTRA overexpression (NTRAOX) and knock-out mutants (ntra-ko) were generated. After exposure to oxidative stress, wild-type and ntra-ko plants were sensitive, but NTRAOX plants tolerant. ROS range was increased by MV in wild-type and ntra-ko plants, but not in NTRAOX. Investigating the involvement of Arabidopsis NTRA in drought, NTRAOX plants exhibited extreme drought tolerance with high survival rates, lower water loss and reduced ROS compared to wild-type and ntra-ko plants. Transcripts of drought-responsive genes, such as RD29A and DREB2A, were highly expressed under drought and antioxidant genes, namely CuZnSOD and APX1 were enhanced in the absence of drought in NTRAOX plants. The results suggest that NTRA overexpression confers oxidative and drought tolerance by regulation of ROS amounts.

Overexpression of chloroplast-localized NADPH-dependent thioredoxin reductase C (NTRC) enhances tolerance to photo-oxidative and drought stresses in Arabidopsis thaliana

Chloroplast is a major organelle that conducts photosynthesis to produce ATP and NADPH via conversion of water to oxygen in plant. While the photosynthesis occurs, molecular oxygen easily changes to reactive oxygen species (ROS) consisting of toxic oxygen radicals resulting in oxidative stress. NADPH-dependent thioredoxin reductases (NTRs) play a pivotal role to regulate the redox state of the thioredoxin system providing reducing power to peroxidase. Here, we identify whether chloroplast NTRC confers stress tolerance through maintenance of ROS in Arabidopsis. NTRC transcripts were two-fold induced at 1 h treatment exposed to a photooxidative agent, methyl viologen (MV). The enhanced NTRC transcripts conferred oxidative stress tolerance displaying that NTRC overexpressing plants (NTRCOX) were tolerant compared to Col-0 and knock-out (ntrc-ko) plants on MVcontaining media. MV-mediated ROS induction was not detected in NTRCOX whereas that was highly accumulated in Col-0 and ntrc-ko. We further examined that NTRCOX showed extreme drought tolerance with lower water loss compared to Col-0 and ntrc-ko. Drought-responsive genes such as RD29A and DREB2A were enhanced in NTRCOX by drought compared to Col-0 and ntrc-ko. The results suggest that NTRC overexpression contributes to maintaining ROS homeostasis under stress conditions and confers the tolerance to photo-oxidative and drought stresses.

Development of in vitro HSP90 foldase chaperone assay using a GST-fused Real-substrate, ZTL (ZEITLUPE)

Protein folding is one of the essential and fundamental processes involved in all living organisms wherein Heat-Shock Protein 90 (HSP90) served as a chaperone which plays important function in protein folding and further promotes refolding of denatured proteins. There are over six hundred identified substrates of HSP90 at present; however, there is a need to develop a specific folding assay method to test its in vitro foldase activity in case of difficulty in substrate activity measurement in vitro. In previous studies, it has been reported that HSP90 is necessary for the stabilization of ZEITLUPE (ZTL) which plays in the circadian clock and photomorphogenesis in Arabidopsis (Kim et al. 2011). Rhythmic oscillations of ZTL protein might be caused by denaturation and stabilization, thus, refolding activity of HSP90 is possibly involved in the oscillations of ZTL protein. However, the question whether HSP90 indeed promotes refolding of ZTL or not has not yet identified and therefore needs to be investigated. Recombinant glutathione-S transferase (GST) fused-ZTL in bacteria was produced and purified as the soluble GST-ZTL through treatments of both sarkosyl and Triton-X100 as ionic and nonionic detergents, respectively. Upon heat (45°C) treatment, the GST activity of GST-ZTL decreased significantly. After the 3 h heat-induced denaturation of GST-ZTL, the refolding of denatured GST-ZTL was enhanced upon addition of HSP90 in a dose-dependent manner. The results from this study showed that recombinant GST-fused protein can be possibly used for in vitro protein refolding assay method. Moreover, results revealed that Arabidopsis HSP90 can efficiently refolds the denatured GST-ZTL in vitro.

Author Correction: The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering

The previously published version of this Article contained errors in Figure 5. In panel c, the second and fourth blot images were incorrectly labeled ‘α-Myc’ and should have been labelled ‘α-HA’. These errors have been corrected in both the PDF and HTML versions of the Article.

Photoperiod sensing system for timing of flowering in plants

CONSTANS (CO) induces the expression of FLOWERING LOCUS T (FT) in the photoperiodic pathway, and thereby regulates the seasonal timing of flowering. CO expression is induced and CO protein is stabilized by FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1) in the late afternoon, while CO is degraded by CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) during the night. These regulatory cascades were thought to act independently. In our study, we investigated the relationship between FKF1 and COP1 in the regulation of CO stability in response to ambient light conditions. A genetic analysis revealed that FKF1 acts as a direct upstream negative regulator of COP1, in which cop1 mutation is epistatic to fkf1 mutation in the photoperiodic regulation of flowering. COP1 activity requires the formation of a hetero-tetramer with SUPPRESSOR OF PHYA-105 (SPA1), [(COP1)2(SPA1)2]. Light-activated FKF1 has an increased binding capacity for COP1, forming a FKF1-COP1 hetero-dimer, and inhibiting COP1 homo-dimerization at its coiled-coil (CC) domain. Mutations in the CC domain result in poor COP1 dimerization and misregulation of photoperiodic floral induction. We propose that FKF1 represses COP1 activity by inhibiting COP1 dimerization in the late afternoon under long-day conditions, resulting in early flowering.