NAC Transcription Factor ORE1 and Senescence-Induced BIFUNCTIONAL NUCLEASE1 (BFN1) Constitute a Regulatory Cascade in Arabidopsis

Abstract

Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoter-reporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.

ETHYLENE-INSENSITIVE3 Is a Senescence-Associated Gene That Accelerates Age-Dependent Leaf Senescence by Directly Repressing miR164 Transcription in Arabidopsis

“…Collectively, these molecular and genetic studies bring about a wholly connected signaling cascade of EIN2-EIN3-miR164-NAC2, which we refer to as the NAC2 pathway. Several studies have identified a subset of downstream genes of NAC2 transcription factor in the control of senescence (Balazadeh et al, 2010), such as BIFUNCTIONAL NUCLEASE1, SAG29/SWEET15, and SINA1 (Matallana- Ramirez et al, 2013). Together with our findings, it is thus possible to assemble the signaling pathways and molecular network that modulate the intricate processes of leaf senescence.…”

Transcription Factor ATAF1 in Arabidopsis Promotes Senescence by Direct Regulation of Key Chloroplast Maintenance and Senescence Transcriptional Cascades

“…After selection, 34 clones with high binding activity were sequenced. The sequence alignment of these clones and base substitution or insertion analysis, together with knowledge about the allowable spacer length of homologous NAC protein-binding sites (Xue et al, 2006;Matallana-Ramirez et al, 2013), revealed two types of bipartite sequences, namely VDHVnnYRR (5-6n)YACGnMWSK (BS1) and RRWnnCGTR(5-6n) DACGTMWHD (BS2), as ATAF1-binding sites (underlined nucleotides represent 100% conservation in the sequenced clones; Supplemental Table S2). Recently, Jensen et al (2013) used protein-binding microarrays covering all possible 8-mer DNA sequences and reported the monopartite 6-mer motifs TT[A,C,G]CGT and T(A,C,G)CGT(A,G) as the binding sites of ATAF1.…”

“…In Arabidopsis, positive senescence regulators include ORESARA1 (ORE1; also called ARABIDOPSIS NAC092 [ANAC092] or AtNAC2; Kim et al, 2009;Balazadeh et al, 2010a;Rauf et al, 2013a), ORESARA1 SISTER1 (ORS1; ANAC059; Balazadeh et al, 2011), AtNAP (for Arabidopsis NAC-LIKE, ACTIVATED BY APETALA3/ PISTILLATA; ANAC029; Guo and Gan, 2006), and ANAC016 , while negative regulators include JUNGBRUNNEN1 (JUB1; ANAC042; Wu et al, 2012) and VND-INTERACTING2 (ANAC083; Yang et al, 2011). Furthermore, downstream target genes of some of the senescence-regulated NAC TFs have been reported, including BIFUNCTIONAL NUCLEASE I (BFN1), RIBONUCLEASE3, SEVEN-IN-ABSENTIA1 (SINA1), and SENESCENCE-ASSOCIATED GENE29 (SAG29; SWEET15) for ORE1 (Balazadeh et al, 2010a;Matallana-Ramirez et al, 2013;Rauf et al, 2013a), DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2 for JUB1 (Wu et al, 2012), and SAG113 for AtNAP (Guo and Gan, 2006). These studies reveal first insights into the complex regulatory networks underlying plant senescence.…”

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis

“…Several stay-green mutants have been isolated, of which many are transcription factor mutants (Kusaba et al, 2013;Penfold and Buchanan-Wollaston, 2014). For example, the NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF1-2], and cup-shaped cotyledon [CUC2]) transcription factor ORESARA1 (ORE1) is regulated at the transcriptional and posttranscriptional levels and promotes leaf senescence through transcriptional modulation of target genes, such as BIFUNCTIONAL NUCLEASE1 and GOLDEN2-LIKE1/2 (Kim et al, 2009;Matallana-Ramirez et al, 2013;Rauf et al, 2013). As mentioned previously, plant hormones play an important role in regulating leaf senescence.…”