Byoung-Doo Lee

STAY-GREEN and Chlorophyll Catabolic Enzymes Interact at Light-Harvesting Complex II for Chlorophyll Detoxification during Leaf Senescence in Arabidopsis

This work shows that the chloroplast-localized components of the chlorophyll catabolic pathway dynamically interact with each other, possibly forming a multiprotein complex specifically localizing to light-harvesting complex II. This interaction likely channels chlorophyll breakdown intermediates and thereby prevents potential chlorophyll-derived phototoxicity during leaf senescence. During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel’s green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing AREB1 Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP

The Arabidopsis transcription factor NAC016 activates drought stress responses by inducing NAP transcription and repressing AREB1 transcription by binding to different regions of the AREB1 promoter. Drought and other abiotic stresses negatively affect plant growth and development and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stress-responsive signaling. Here, we show that Arabidopsis thaliana NAC016 is involved in drought stress responses; nac016 mutants have high drought tolerance, and NAC016-overexpressing (NAC016-OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]CA, in the promoters of genes downregulated in nac016-1 mutants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or its reverse complement CGTG). NAC016 directly binds to the NAC16BM in the promoter of ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses AREB1 transcription. We found that knockout mutants of the NAC016 target gene NAC-LIKE, ACTIVATED BY AP3/PI (NAP) also exhibited strong drought tolerance; moreover, NAP binds to the AREB1 promoter and suppresses AREB1 transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving NAC016, NAP, and AREB1 functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis.

Rice FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1) promotes flowering independent of photoperiod.

In the facultative long-day (LD) plant Arabidopsis thaliana, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is activated by blue light and promotes flowering through the transcriptional and post-translational regulation of CONSTANS under inductive LD conditions. By contrast, the facultative short day (SD) plant rice (Oryza sativa) flowers early under inductive SD and late under non-inductive LD conditions; the regulatory function of OsFKF1 remains elusive. Here we show that osfkf1 mutants flower late under SD, LD and natural LD conditions. Transcriptional analysis revealed that OsFKF1 up-regulates the expression of the floral activator Ehd2 and down-regulates the expression of the floral repressor Ghd7; these regulators up- and down-regulate Ehd1 expression, respectively. Moreover, OsFKF1 can up-regulate Ehd1 expression under blue light treatment, without affecting the expression of Ehd2 and Ghd7. In contrast to the LD-specific floral activator Arabidopsis FKF1, OsFKF1 likely acts as an autonomous floral activator because it promotes flowering independent of photoperiod, probably via its distinct roles in controlling the expression of rice-specific genes including Ehd2, Ghd7 and Ehd1. Like Arabidopsis FKF1, which interacts with GI and CDF1, OsFKF1 also interacts with OsGI and OsCDF1 (also termed OsDOF12). Thus, we have identified similar and distinct roles of FKF1 in Arabidopsis and rice.

Tobacco phytochelatin synthase (NtPCS1) plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins, which are involved in heavy metal detoxification in plants and other living organisms. Previously, we cloned a PCS1 gene from tobacco (Nicotiana tabacum) and showed that its expression in yeast (Saccharomyces cerevisiae) resulted in increased cadmium (Cd) tolerance and Cd accumulation (Kim et al., J Plant Biol 48:440–447, 2005). To examine the role of NtPCS1 in tobacco, we generated transgenic tobacco lines over-expressing NtPCS1 in the sense or antisense direction. Compared with other PCS1-expressing plants, NtPCS1-expressing tobacco exhibited a unique phenotype: increased tolerance to cadmium and arsenite, but no change in cadmium and arsenic accumulation. In addition, the antisense-NtPCS1 tobacco lines showed growth retardation in the early stage, suggesting that phytochelatin also plays a role in plant development. These results demonstrate that NtPCS1 plays important roles in metal(loid) tolerance as well as in growth and development in tobacco.

Overexpression of NtUBQ2 encoding Ub-extension protein enhances cadmium tolerance by activating 20S and 26S proteasome in tobacco (Nicotiana tabacum)

The Ub/26S proteasome system removes abnormal proteins and most short-lived regulatory proteins, thereby contributing to cell proliferation, hormone responses, development and resistance to abiotic and biotic stresses. Here we show that cadmium tolerance is related positively to the 20S proteasome (catalytic particle of 26S proteasome) activity in plants. By transforming WT yeast Y800 with a tobacco expression cDNA library, we isolated a tobacco cDNAs, NtUBQ2 (the Ub-extension protein) conferring cadmium tolerance. Overexpression of NtUBQ2 increased cadmium tolerance in transgenic tobacco; 20S proteasome activity was enhanced and ubiquitinated protein level was diminished in response to cadmium. In contrast, proteasome activity was reduced and ubiquitinated protein level was less decreased than transgenic tobacco by Cd treatment in control tobacco which is sensitive to Cd. These observations strongly suggest that plants acquire cadmium tolerance by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or Ub-independent 20S proteasome. This finding could be applied to engineering efficient metal phytoremediators.

Negative regulatory roles of DE-ETIOLATED1 in flowering time in Arabidopsis

Arabidopsis flowers early under long days (LD) and late under short days (SD). The repressor of photomorphogenesis DE-ETIOLATED1 (DET1) delays flowering; det1-1 mutants flower early, especially under SD, but the molecular mechanism of DET1 regulation remains unknown. Here we examine the regulatory function of DET1 in repression of flowering. Under SD, the det1-1 mutation causes daytime expression of FKF1 and CO; however, their altered expression has only a small effect on early flowering in det1-1 mutants. Notably, DET1 interacts with GI and binding of GI to the FT promoter increases in det1-1 mutants, suggesting that DET1 mainly restricts GI function, directly promoting FT expression independent of CO expression. Moreover, DET1 interacts with MSI4/FVE, which epigenetically inhibits FLC expression, indicating that the lack of FLC expression in det1-1 mutants likely involves altered histone modifications at the FLC locus. These data demonstrate that DET1 acts in both photoperiod and autonomous pathways to inhibit expression of FT and SOC1. Consistent with this, the early flowering of det1-1 mutants disappears completely in the ft-1 soc1-2 double mutant background. Thus, we propose that DET1 is a strong repressor of flowering and has a pivotal role in maintaining photoperiod sensitivity in the regulation of flowering time.

The tobacco gene Ntcyc07 confers arsenite tolerance in Saccharomyces cerevisiae by reducing the steady state levels of intracellular arsenic

We cloned a plant gene, Ntcyc07, conferring arsenite tolerance by expressing a tobacco expression library in WT yeast (Y800). Expression of Ntcyc07 increased the tolerance to As(III) and decreased its accumulation, suggesting that the enhanced As(III) tolerance resulted from a reduction of the intracellular arsenic level. Interestingly, expression of Ntcyc07 increased the expression of the As(III) export carrier ACR3, but repressed that of As(III) uptake channel FPS1. Ntcyc07p interacted with Acr1p, which is the transcriptional activator of ACR3, but not with the ACR3 promoter. Taken together, the data indicated that Ntcyc07p promoted As(III) tolerance by decreasing the intracellular level of As(III) via increasing the expression of ACR3 and reducing that of FPS1.

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.