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>

Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Sakuraba, Y.,Park, S.Y.,Kim, Y.S.,Wang, S.H.,Yoo, S.C.,Hortensteiner, S.,Paek, N.C. Oxford University Press 2014 Molecular plant Vol.7 No.8

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.

Arabidopsis STAYGREEN-LIKE (SGRL) promotes abiotic stress-induced leaf yellowing during vegetative growth

Sakuraba, Y.,Kim, D.,Kim, Y.S.,Hortensteiner, S.,Paek, N.C. North-Holland Pub ; Elsevier Science Ltd 2014 FEBS letters Vol.588 No.21

During leaf senescence in Arabidopsis, STAYGREEN 1 (SGR1) and SGR2 regulate chlorophyll degradation positively and negatively, respectively. SGR-LIKE (SGRL) is also expressed in pre-senescing leaves, but its function remains largely unknown. Here we show that under abiotic stress, Arabidopsis plants overexpressing SGRL exhibit early leaf yellowing and sgrl-1 mutants exhibit persistent green color of leaves. Under salt stress, SGR1 and SGRL act synergistically for rapid Chl degradation prior to senescence. Furthermore, SGRL forms homo- and heterodimers with SGR1 and SGR2 in vivo, and interacts with LHCII and chlorophyll catabolic enzymes. The role of SGRL under abiotic stress is discussed.

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

Sakuraba, Y.,Schelbert, S.,Park, S.-Y.,Han, S.-H.,Lee, B.-D.,Andres, C.B.,Kessler, F.,Hortensteiner, S.,Paek, N.-C. AMERICAN SOCIETY OF PLANT BIOLOGISTS 2012 The Plant cell Vol.24 No.2

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>

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.

Arabidopsis NAC016 promotes chlorophyll breakdown by directly upregulating STAYGREEN1 transcription

Sakuraba, Y.,Han, S. H.,Lee, S. H.,Hö,rtensteiner, S.,Paek, N. C. Springer International 2016 Plant cell reports Vol. No.

<P>During leaf senescence or abiotic stress in Arabidopsis thaliana, STAYGREEN1 (SGR1) promotes chlorophyll (Chl) degradation, acting with Chl catabolic enzymes, but the mechanism regulating SGR1 transcription remains largely unknown. Here, we show that the Arabidopsis senescence-associated NAC transcription factor NAC016 directly activates SGR1 transcription. Under senescence-promoting conditions, the expression of SGR1 was downregulated in nac016-1 mutants and upregulated in NAC016-overexpressing (NAC016-OX) plants. By yeast one-hybrid and chromatin immunoprecipitation assays, we found that NAC016 directly binds to the SGR1 promoter, which contains the NAC016-specific binding motif (termed the NAC016BM). Furthermore, nac016-1 SGR1-OX plants showed an early leaf yellowing phenotype, similar to SGR1-OX plants, confirming that NAC016 directly activates SGR1 expression in the leaf senescence regulatory cascade. Although we found that NAC016 activates SGR1 expression in senescing leaves, this transcriptional regulation is considerably weaker in maturing seeds; the seeds of sgr1-1 mutants (also known as nonyellowing1-1, nye1-1) stayed green, while the seeds of nac016-1 mutants turned from green to yellow normally. We also found that the abscisic acid (ABA) signaling-related transcription factor genes ABI5 and EEL and the ABA biosynthesis gene AAO3, which activate SGR1 expression directly or indirectly, were significantly downregulated in nac016-1 mutants and upregulated in NAC016-OX plants. However, the NAC016BM does not exist in their promoter regions, indicating that NAC016 may indirectly activate these ABA signaling and biosynthesis genes, probably by directly activating transcriptional cascades regulated by the NAC transcription factor NAP. The NAC016-mediated regulatory cascades of SGR1 and other Chl degradation-related genes are discussed.</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>

Mutation of Rice Early Flowering3.1 (OsELF3.1) delays leaf senescence in rice

Sakuraba, Y.,Han, S. H.,Yang, H. J.,Piao, W.,Paek, N. C. Springer Science + Business Media 2016 Plant Molecular Biology Vol. No.

<P>In Arabidopsis, EARLY FLOWERING3 (ELF3) has pivotal roles in controlling circadian rhythm and photoperiodic flowering. In addition, ELF3 negatively regulates leaf senescence by repressing the transcription of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR5 (PIF5); elf3 mutants senesce earlier and ELF3-overexpressing (ELF3-OX) plants senesce later than wild type (WT). Here, we show that in contrast to Arabidopsis ELF3, which represses senescence, the rice homolog OsELF3.1 promotes leaf senescence; oself3.1 mutants showed delayed senescence and OsELF3.1-OX plants senesced earlier under both dark-induced and natural senescence conditions. Microarray analysis revealed that in the senescing leaves, a number of senescence-associated genes, phytohormone-related genes, and NAC and WRKY family genes (OsNAP, ONAC106, and OsWRKY42) were differentially expressed in oself3.1 mutants compared with WT. Interestingly, we found that Arabidopsis plants overexpressing OsELF3.1 show delayed leaf senescence, produce short petioles, and flower late in long days, just like Arabidopsis ELF3-OX plants. This demonstrates that the regulatory functions of ELF3 and OsELF3.1 are conserved between Arabidopsis and rice, but the downstream regulatory cascades have opposite effects.</P>