Sub-Group III Networks: From Stress Responses to Senescence
The first indications of a NAC-centered GRN came from the identification of ANAC019, ANAC055, and ANAC072 as direct regulators of ERD1, which encodes a Clp protease regulatory subunit (Fig. 3d) . Overexpression of one of the three NAC genes is not sufficient for up regulation of ERD1 , but depends on co-expression with the zinc finger homeodomain TF ZFHD1. ZFHD1 also recognizes a cis-acting element in the ERD1 promoter and is up-regulated by drought, high salt, and ABA . This suggests cooperative regulation of stress responses by different TF families in drought tolerance (Fig. 3a) through physical interaction between the NAC and the ZFHD1 TFs . Microarray analysis of transgenic plants over expressing one of the three NAC TFs revealed up-regulation of the expression of several stress and ABA-inducible genes including RD29B, COR47, ERD11, and FER1 genes [1, 7] further supporting the role of these TFs in abiotic stress signaling.
Figure 3. Gene regulatory and protein–protein interaction networks of NAC TFs in stress responses and senescence. Direct interactions between the NAC TFs and DNA are shown by arrows (positive effect on expression) or blocked lines (negative effect on expression). Protein–protein interactions are shown by straight lines, and agonistic effects of these interactions are shown by arrows, whereas antagonistic effects are shown by blocked lines. Dashed arrows and lines refer to effects which may involve additional components. (a) Sub-group III-3 ANAC019, ANAC055, and ANAC072 GRN main associations are: MYC2 with JA-signaling and pathogen defense; MYBs, ABFs, and ABI with ABA-signaling and abiotic stress responses, CBF1, 2, and 3 with cold responses, and CBF4 also with ABA. Up-regulation of sub-group III-II ANAC019, ANAC055, and ANAC072 improves drought tolerance either directly or through interactions with ZFHD1. (b) Sub-group III-3 ATAF1 GRN. (c) Sub-group III-3 ATF2 GRN. (d) Sub-group III-2 AtNAP GRN. (e) Sub-group III-2 VNI1 GRN. (f) Sub-group V-1 JUB1 GRN. (g) Sub-group II-3 ORE1 GRN. (h) Sub-group IV-1 ANAC096 GRN. (i) Sub-group I-1 NTL6 GRN. (j) Sub-group I-2 NTL4 GRN. (k) Sub-group I-1 CBNAC GRN.
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The first direct up-stream regulators of these three NAC TFs were recently identified in a yeast one-hybrid screen . The key ABA-regulating bZIP TFs, ABA RESPONSIVE ELEMENT BINDING FACTOR3 (ABF3), and ABF4 , were identified by their ability to bind to NAC promoter fragments, in accordance with the ABA-association of the three NAC TFs [1, 7, 47]. ANAC019 and ANAC055 also interact with another key mediator of ABA-signaling, ABI4, an AP2 family TF . Five members of the MYB TF family, MYB2, MYB21, MYB108, MYB112, and MYB116 also bound to the promoter fragments. In contrast, the four AP2 TFs, C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT BINDING FACTOR1 (CBF1), CBF2, CBF3, and CBF4 bound to only the ANAC072 promoter. Modeling of expression and mutant analysis suggested further differential contribution to the regulation of the expression of the three NAC genes by potential regulators in stress responses and senescence. For example, of the MYB TFs which bind to the promoters of all three NAC TFs, only MYB2 and MYB108 are implicated in senescence, and although all four CBF TFs can regulate ANAC072, they may play different roles in binding under cold, osmotic and salt stresses, senescence, and during Botrytis cinerea infection .
Biotic stress GRNs are also appearing for the three NAC TFs. ANAC019 and ANAC055 are implicated in JA-dependent defense responses with their MeJA-induced gene expression depending on the JA-signaling components CORONTATINE INSENSITIVE1 (COI1) and the basic helix–loop–helix TF AtMYC2. In addition, over expression of ANA019 partially rescues the phenotype of the atmyc2-2 mutant . The phenotype of the anac019anac055 double mutant, which exhibits decreased expression of the JA marker genes LIPOXYGENASE 2 (LOX2) and VSP1 (a direct ANAC019 target), also shows increased resistance to the necrotrophic pathogen B. cinerea . Recently, ChIP was used to show that ANAC019, ANAC055, and ANAC072 are direct in vivo targets of MYC2 . Coronatine, a toxin produced by the pathogenic bacterium Pseudonomas syringae and mimicking JA isoleucine, activates the three NAC genes. The NAC TFs mediate coronatine-induced stomatal reopening and bacterial propagation by inhibiting accumulation of the immune signaling hormone salicylic acid (SA), thereby promoting pathogen virulence. The NAC TFs exert this inhibitory effect by binding (at least in the case of ANAC019) to and repressing ISOCHORISMATE SYNTHASE1 (ICS1) and by activating S-ADENOSYLMETHIONINE-DEPENDENT METHYLTRANSFERASE (BSMT1) involved in SA biosynthesis and metabolism, respectively. Together these studies elucidate the complexity and intersections of the GRNs of ANAC019, ANAC055, and ANAC072 involving ABA- and JA-signaling and enhancing and repressing adaptive abiotic and biotic stress responses as well as developmental senescence signaling (Fig. 3a).
ANAC019 was originally identified as an interaction partner of the small RING-H2 protein RHA2a , a positive regulator of ABA-signaling, and a functional E3 ubiquitin ligase . Additional RING-H2 proteins also interact with ANAC019 in yeast two-hybrid assays (Fig. 3a) . However, it remains to be determined if these proteins regulate ANAC019 by ubiquitin-dependent degradation. A putative function of the interaction between ANAC019 and CTD phosphate-like1 (CPL1), which is essential for regulation of stress-responsive gene expression, also remains elusive . Thus, a functional role has so far only been revealed for the interaction with ZFHD1. The NAC DBD has been shown to mediate the interaction with RHA2a and ZFHD1 (Fig. 1c), demonstrating the ability of this domain to interact with molecules other than DNA. Clearly, additional interaction partners, also targeting the disordered TRDs, can be expected.
ATAF1 targets the ABA-biosynthesis gene NCED3 in vivo, and ATAF1-mediated induction of NCED3 expression correlates with increased ABA levels (Fig. 3b) . Plants over-expressing ATAF1 have reduced stomatal aperture , and transient expression of NCED3 in guard cells also decreases stomatal aperture  suggestive of overlapping physiological functions of ATAF1 and NCED3. Thus, ATAF1 may regulate ABA biosynthesis in a positive feed-back loop by binding to and activating the expression of NCED3. Plants over-expressing ATAF1 display stunted growth and delayed flowering, which is also the case for ABA-hypersensitive plants over-expressing the ATAF1 interaction partner kinase SnRK1.1/AKIN10 [53, 54]. This interaction is likely to involve the relatively disordered TRD of ATAF1  (Fig. 1c). Future studies should address the functional and physiological consequences of this interaction in planta. However, ATAF1 seems largely dispensable for SnRK1 signaling as ataf1 mutant plants display wild-type phenotypes . In addition to NCED3, several other putative ATAF1 targets show ATAF1-dependent expression perturbations. This is the case for ABI2, which as a typical clade A type 2C protein phosphate (PP2C) functions as a negative regulator of ABA signaling by dephosphorylating ABA-activated kinases . Although speculative, the positive correlation between ATAF1 and ABI2 may reflect a negative feed-back loop to decrease endogenous ABA levels associated with ATAF1 induction to limit accelerated ABA signaling and thereby physiological defects .
ATAF2, closely related to ATAF1, is also induced by stresses (Fig. 3c) . Recently, ATAF2 was shown to be involved in auxin hormone biosynthesis via regulation of NIT2  encoding an enzyme converting indole-3-3acetonitril (IAN) into auxin in a pathway that can be activated by wounding and pathogens . Expression of both ATAF2 and NIT2 was induced by IAN treatment, and ATAF2 activates the promoter of the NIT2 genes by direct binding. Further studies are needed to clarify how ATAF2 contributes to tryptophan-dependent auxin biosynthesis, which can be activated by wounding and pathogens, and if this is related to plant immunity .
The involvement of group III NAC TFs in senescence is clear from analysis of AtNAP (Fig. 3d). A T-DNA insertion knockout of AtNAP displays a delay in leaf senescence, and overexpression of AtNAP in young leaves causes precocious senescence suggesting that AtNAP is a positive regulator of senescence . SAG113 is a direct target of AtNAP , and both AtNAP and SAG113 are induced by leaf senescence and ABA. SAG113, which encodes a Golgi-localized protein phosphatase 2C family protein phosphatase, mediates inhibition of stomatal closure resulting in water loss, and triggering senescence in leaves [30, 57].
The expression of VNI2 is induced by senescence and by salt in an ABA-dependent manner, and overexpression of VNI2 enhances resistance to salt stress and prolongs leaf longevity, similarly to the direct target genes COR15A, COR15B, RD29A, and RD29B (Fig. 1e) . Thus, VNI2 integrates ABA-mediated abiotic stress signals and ageing. VNI2 is also an interaction partner of VASCULAR-RELATED NAC-DOMAIN7 (VND7) associated with programmed cell death (PCD) , suggesting that abiotic stress adaptation, senescence, and PCD are integrated via a VIN2 centered GRN. In conclusion, several group III NAC TFs are convergence points for different signaling pathways related to stress and senescence, and regulated by several different phytohormones.
As a key senescence-control TF ORE1 directly targets the BFN1, SAG29/SWEET15, and SINA1 genes , relating ORE1 to different physiological functions (Fig. 3g). ORE1 and BFN1 have overlapping expression patterns, and ANAC092 and BFN1 were suggested to constitute a regulatory cascade resulting in nucleic acid degradation during senescence and PCD . SAG29, encoding a putative sugar transporter, accelerates senescence when overexpressed , possibly by altering sugar transport, and SINA1, encoding a putative E3 ubiquitin ligase, may mediate protein ubiquitination and degradation. ORE1 itself is controlled by mRNA degradation by the transacting micro(mi)RNA164 . miR164 in turn is negatively regulated both by ETHYLENE INSENSITIVE 2 (EIN2), central to ethylene signaling , and by its downstream target gene EIN3, encoding a key TF in ethylene signaling which directly represses miR164 accumulation . This again allows ORE1 mRNA accumulation .
The Golden2-like TFs, GLK1 and GLK2, were recently shown to interact with the ORE1 TRD  (Figs. 1c and 3f). GLK1 and GLK2 are important for chloroplast development and maintenance and thereby for leaf maintenance . Whereas the interaction does not affect the transactivating ability of ORE1, it antagonizes GLK transcriptional activity. A model was proposed in which ORE1 accumulation and interaction with the GLKs at late stages of senescence result in ORE1-GLK heterodimers incapable of activating photosynthetic GLK target genes . This regulatory interaction reveals a novel mechanism for fine tuning senescence and demonstrates the functional power of the ORE1 TRD in protein–protein interactions.
ANAC096: A Novel Player in Intersecting Abiotic Stress GRNs
The involvement of ANAC096 in ABA-mediated stress responses was suggested from a screen for mutants with ABA-hyposensitive phenotypes (Fig. 3h) . ANAC096 functions as a positive regulator of ABA-responsive signaling under dehydration and osmotic stress, and a major proportion of ABA-responsive genes are transcriptionally regulated by ANAC096. It directly interacts with ABF2 and ABF4 (Fig. 1c), and ANAC096 and ABF2 synergistically activate RD29A transcription. Furthermore, the anac096 abf2 abf4 triple mutant is more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. ANAC096 was therefore suggested to synergistically activate, with the ABFs, ABA-inducible genes in response to dehydration and osmotic stress . This is one of several examples of NAC TF interplay with other types of TFs which may be essential for rapid and specific regulation of gene expression upon stress exposure. In addition to enhancing specificity, such mechanisms allow synergistic effects from different pathways involved in different functions.
Interestingly, ANAC096 interacts with ABF2 and ABF4 but not with ABF3 . The explanation for this differentiation of specificity remains elusive. The fact that ANAC096 has an agonistic, and ANAC019 an antagonistic relationship with ABF2 with respect to RD29A expression, also raises a biochemical question. As the NAC domains are highly conserved , ANAC096 and ANAC019 may use their mostly disordered and variable TRDs for the interaction with the ABFs . This region of ANAC096 has also been suggested to be responsible for the interaction with the corepressor protein TOPLESS (TPL) .
Networks of Membrane-Anchored NAC TFs
The plasma membrane-anchored NTL6 of sub-group I-1 is proteolytically processed upon exposure to cold and migrates to the nucleus, where it induces the expression of the direct target genes PR1, PR2, and PR5, indicating that NTL6 mediates cold-induced pathogenesis (Fig. 3i) . In addition, ABA promotes NLT6 processing, and transgenic plants overexpressing processed NTL6 are hypersensitive to ABA and high salinity . Taken together, these observations indicate that NTL6 integrates plant responses to both biotic and abiotic stress conditions . It was recently shown that Snf1-related protein kinase (SnRK) 2.8-mediated phosphorylation of NTL6 is necessary for nuclear import of NTL6 . Overexpression of NTL6 results in enhanced drought resistance, as does overexpression of SnRK2.8, and SnRK2.8 knock-out compromises the effect of NTL6 overexpression. This suggests that the SnRK2.8-mediated phosphorylation of NTL6 is important for the NTL6-mediated induction of drought-resistance .
The ABA, drought, and heat inducible NTL4 gene encodes a sub-group I-2 membrane-anchored NAC TF which promotes ROS production during drought-induced senescence in Arabidopsis (Fig. 3j). NTL4 directly up-regulates the Atrboh (A, C, and E) genes encoding ROS biosynthetic proteins . Leaf senescence is accelerated in transgenic plants overexpressing proteolytically processed NTL4 under drought conditions, and these transgenics are hypersensitive to drought and ROS accumulates in their leaves. These observations indicate that NTL4 regulates ROS generation as part of processes controlling drought-induced leaf senescence in Arabidopsis . In conclusion, membrane-anchored NTL TFs are nodes connecting different signaling pathways of abiotic and biotic stress responses and of abiotic stress responses and senescence.
An interesting mechanism of suppression of basal pathogen resistance has been reported for CBNAC/NTL9 whose membrane anchoring is debated (Fig. 3k) [33, 68]. cbnac1 mutants display enhanced resistance to the bacterial pathogen P. syringae (Pst) through relief of PR1 repression. SUPPRESSOR OF NONEXPRESSOR OF PR GENES1, INDUCIBLE1 (SNI1), also a negative regulator of PR1 expression, interacts with CBNAC and enhances binding of CBNAC to the PR1 promoter. Thus, CBNAC and SNI1 could function synergistically as negative regulators of PR1 expression and disease resistance to prevent detrimental effects of constitutive activation of defense on plant growth , or they could be Pst effector targets guarded by specific Arabidopsis R genes. The C-terminal of the NAC TF is also used for their interaction in this case (Fig. 1c).