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

Sakuraba, Yasuhito,Kim, Ye-Sol,Han, Su-Hyun,Lee, Byoung-Doo,Paek, Nam-Chon American Society of Plant Biologists 2015 The Plant cell Vol.27 No.6

<P>The Arabidopsis transcription factor NAC016 activates drought stress responses by inducing <I>NAP</I> transcription and repressing <I>AREB1</I> transcription by binding to different regions of the <I>AREB1</I> promoter.</P><P>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 <I>Arabidopsis thaliana</I> NAC016 is involved in drought stress responses; <I>nac016</I> mutants have high drought tolerance, and <I>NAC016</I>-overexpressing (<I>NAC016</I>-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 <I>nac016-1</I> 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 <I>ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1</I> (<I>AREB1</I>), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses <I>AREB1</I> transcription. We found that knockout mutants of the NAC016 target gene <I>NAC-LIKE, ACTIVATED BY AP3/PI</I> (<I>NAP</I>) also exhibited strong drought tolerance; moreover, NAP binds to the <I>AREB1</I> promoter and suppresses <I>AREB1</I> transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving <I>NAC016</I>, <I>NAP</I>, and <I>AREB1</I> functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis.</P>

Roles of rice PHYTOCHROME-INTERACTING FACTOR-LIKE1 (OsPIL1) in leaf senescence

Sakuraba, Yasuhito,Kim, Eun-Young,Paek, Nam-Chon Informa UK (Taylor Francis) 2017 Plant signaling & behavior Vol.12 No.9

Rice Phytochrome-Interacting Factor-Like1 (OsPIL1) is involved in the promotion of chlorophyll biosynthesis through feed-forward regulatory loops

Sakuraba, Yasuhito,Kim, Eun-Young,Han, Su-Hyun,Piao, Weilan,An, Gynheung,Todaka, Daisuke,Yamaguchi-Shinozaki, Kazuko,Paek, Nam-Chon Oxford University Press 2017 Journal of experimental botany Vol.68 No.15

<▼1><P>In the tightly regulated chlorophyll biosynthesis pathway, the transcription factor Rice Phytochrome-Interacting Factor-Like1 promotes chlorophyll biosynthesis by up-regulating chlorophyll biosynthetic genes through feed-forward regulatory loops involving <I>GOLDEN2-LIKE1</I> (<I>OsGLK1</I>) and <I>OsGLK2</I>.</P></▼1><▼2><P><B>Abstract</B></P><P>In phototrophic plants, the highly conserved and tightly regulated process of chlorophyll (Chl) biosynthesis comprises multi-step reactions involving more than 15 enzymes. Since the efficiency of Chl biosynthesis strongly affects plant productivity, understanding the underlying regulatory mechanisms in crop plants can be useful for strategies to increase grain and biomass yields. Here, we show that rice (<I>Oryza sativa</I>) Phytochrome-Interacting Factor-Like1 (OsPIL1), a basic helix-loop-helix transcription factor, promotes Chl biosynthesis. The T-DNA insertion knockdown <I>ospil1</I> mutant showed a pale-green phenotype when grown in a natural paddy field. Transcriptome analysis revealed that several genes responsible for Chl biosynthesis and photosynthesis were significantly down-regulated in <I>ospil1</I> leaves. Using promoter binding and transactivation assays, we found that OsPIL1 binds to the promoters of two Chl biosynthetic genes, <I>OsPORB</I> and <I>OsCAO1</I>, and promotes their transcription. In addition, OsPIL1 directly up-regulates the expression of two transcription factor genes, <I>GOLDEN2-LIKE1</I> (<I>OsGLK1</I>) and <I>OsGLK2</I>. OsGLK1 and OsGLK2 both bind to the promoters of <I>OsPORB</I> and <I>OsCAO1</I>, as well as some of genes encoding the light-harvesting complex of photosystems, probably promoting their transcription. Thus, OsPIL1 is involved in the promotion of Chl biosynthesis by up-regulating the transcription of <I>OsPORB</I> and <I>OsCAO1</I> via trifurcate feed-forward regulatory loops involving two OsGLKs.</P></▼2>

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Sakuraba, Yasuhito,Park, So-Yon,Paek, Nam-Chon Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.5

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a staygreen phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Yasuhito Sakuraba,박소연,백남천 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.5

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a staygreen phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.

Delayed degradation of chlorophylls and photosynthetic proteins in <i>Arabidopsis</i> autophagy mutants during stress-induced leaf yellowing

Sakuraba, Yasuhito,Lee, Sang-Hwa,Kim, Ye-Sol,Park, Ohkmae K.,Hö,rtensteiner, Stefan,Paek, Nam-Chon Oxford University Press 2014 Journal of experimental botany Vol.65 No.14

<P>Plant autophagy, one of the essential proteolysis systems, balances proteome and nutrient levels in cells of the whole plant. Autophagy has been studied by analysing <I>Arabidopsis thaliana</I> autophagy-defective <I>atg</I> mutants, but the relationship between autophagy and chlorophyll (Chl) breakdown during stress-induced leaf yellowing remains unclear. During natural senescence or under abiotic-stress conditions, extensive cell death and early yellowing occurs in the leaves of <I>atg</I> mutants. A new finding is revealed that <I>atg5</I> and <I>atg7</I> mutants exhibit a functional stay-green phenotype under mild abiotic-stress conditions, but leaf yellowing proceeds normally in wild-type leaves under these conditions. Under mild salt stress, <I>atg5</I> leaves retained high levels of Chls and all photosystem proteins and maintained a normal chloroplast structure. Furthermore, a double mutant of <I>atg5</I> and non-functional stay-green <I>nonyellowing1-1</I> (<I>atg5 nye1-1</I>) showed a much stronger stay-green phenotype than either single mutant. Taking these results together, it is proposed that autophagy functions in the non-selective catabolism of Chls and photosynthetic proteins during stress-induced leaf yellowing, in addition to the selective degradation of Chl–apoprotein complexes in the chloroplasts through the senescence-induced STAY-GREEN1/NYE1 and Chl catabolic enzymes.</P>

Mutation of SPOTTED LEAF3 (SPL3) impairs abscisic acid-responsive signaling and delays leaf senescence in rice

Seung-Hyun Wang,Jung-Hyun Lim,Yasuhito Sakuraba,Nam-Chon Paek 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Lesion mimic mutants commonly display spontaneous cell death in pre-senescent green leaves under normal conditions, without pathogen attack. Despite molecular and phenotypic characterization of several lesion mimic mutants, the mechanisms of the spontaneous formation of cell death lesions remain largely unknown. Here, we examined the rice lesion mimic mutant spotted leaf3 (spl3). In mutants grown under a light/dark cycle, spl3 mutants appeared similar to wild type at early developmental stages, but lesions gradually appeared in the mature leaves close to heading stage. By contrast, in mutants grown under continuous light, severe cell death lesions formed in developing leaves, even at the seedling stage. Histochemical analysis showed that hydrogen peroxide accumulated in the mutants, likely causing the cell death phenotype. By map-based cloning and complementation, we showed that a 1-bp deletion in the first exon of Oryza sativa Mitogen-Activated Protein Kinase Kinase Kinase1 (OsMAPKKK1)/OsEDR1/ OsACDR1 causes the spl3 mutant phenotype. We found that the spl3 mutants were insensitive to abscisic acid (ABA), showing normal root growth in ABA-containing media and delayed leaf yellowing during dark-induced and natural senescence. Expression of ABA signaling-associated genes was also less responsive to ABA treatment in the mutants. Furthermore, the spl3 mutants had lower transcript levels and activities of catalases, which scavenge hydrogen peroxide, probably due to impairment of ABA-responsive signaling. Finally we discuss a possible molecular mechanism of lesion formation in the mature leaves of spl3 mutants.

Leaf variegation in the rice zebra2 mutant is caused by photoperiodic accumulation of tetra-cis-lycopene and singlet oxygen

Su-Hyun Han,Choon-Tak Kwon,Yasuhito Sakuraba,Nam-Chon Paek 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O2-], hydrogen peroxide [H2O2], and singlet oxygen [1O2]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during the light period, which consequently altered the expression of 1O2-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive 1O2 under natural day-night conditions.

Transgenic expression of rice MYB102 (OsMYB102) delays leaf senescence and decreases abiotic stress tolerance in Arabidopsis thaliana

( Weilan Piao ),( Yasuhito Sakuraba ),( Nam-chon Paek ) 생화학분자생물학회(구 한국생화학분자생물학회) 2019 BMB Reports Vol.52 No.11

MYB-type transcription factors (TFs) play important roles in plant growth and development, and in the rapid responses to unfavorable environmental conditions. We recently reported the isolation and characterization of a rice (Oryza sativa) MYB TF, OsMYB102, which is involved in the regulation of leaf senescence by downregulating abscisic acid (ABA) biosynthesis and the downstream signaling response. Based on the similarities of their sequences and expression patterns, OsMYB102 appears to be a homolog of the Arabidopsis thaliana AtMYB44 TF. Since AtMYB44 is a key regulator of leaf senescence and abiotic stress responses, it is important to examine whether AtMYB44 homologs in other plants also act similarly. Here, we generated transgenic Arabidopsis plants expressing OsMYB102 (OsMYB102-OX). The OsMYB102-OX plants showed a delayed senescence phenotype during dark incubation and were more susceptible to salt and drought stresses, considerably similar to Arabidopsis plants overexpressing AtMYB44. Real-time quantitative PCR (RT-qPCR) revealed that, in addition to known senescence-associated genes, genes encoding the ABA catabolic enzymes AtCYP707A3 and AtCYP707A4 were also significantly upregulated in OsMYB102- OX, leading to a significant decrease in ABA accumulation. Furthermore, protoplast transient expression and chromatin immunoprecipitation assays revealed that OsMYB102 directly activated AtCYP707A3 expression. Based on our findings, it is probable that the regulatory functions of AtMYB44 homologs in plants are highly conserved and they have vital roles in leaf senescence and the abiotic stress responses. [BMB Reports 2019; 52(11): 653-658]

Rice 7-Hydroxymethyl Chlorophyll a Reductase Is Involved in the Promotion of Chlorophyll Degradation and Modulates Cell Death Signaling

Weilan Piao,한수현,Yasuhito Sakuraba,백남천 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.10

The loss of green coloration via chlorophyll (Chl) degradation typically occurs during leaf senescence. To date, many Chl catabolic enzymes have been identified and shown to interact with light harvesting complex II to form a Chl degradation complex in senescing chloroplasts; this complex might metabolically channel phototoxic Chl catabolic intermediates to prevent oxidative damage to cells. The Chl catabolic enzyme 7-hydroxymethyl Chl a reductase (HCAR) converts 7-hydroxymethyl Chl a (7-HMC a) to Chl a. The rice (Oryza sativa) genome contains a single HCAR homolog (OsHCAR), but its exact role remains unknown. Here, we show that an oshcar knockout mutant exhibits persistent green leaves during both dark-induced and natural senescence, and accumulates 7-HMC a and pheophorbide a (Pheo a) in green leaf blades. Interestingly, both rice and Arabidopsis hcar mutants exhibit severe cell death at the vegetative stage; this cell death largely occurs in a light intensity-dependent manner. In addition, 7-HMC a treatment led to the generation of singlet oxygen (1O2) in Arabidopsis and rice protoplasts in the light. Under herbicide-induced oxidative stress conditions, leaf necrosis was more severe in hcar plants than in wild type, and HCAR-overexpressing plants were more tolerant to re-active oxygen species than wild type. Therefore, in addition to functioning in the conversion of 7-HMC a to Chl a in senescent leaves, HCAR may play a critical role in protecting plants from high light-induced damage by preventing the accumu-lation of 7-HMC a and Pheo a in developing and mature leaves at the vegetative stage.