The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/ hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Lateral root formation in Arabidopsis thaliana is regulated by two related AUXIN RESPONSE FACTORs, ARF7 and ARF19, which are transcriptional activators of early auxin response genes. The arf7 arf19 double knockout mutant is severely impaired in lateral root formation. Target-gene analysis in arf7 arf19 transgenic plants harboring inducible forms of ARF7 and ARF19 revealed that ARF7 and ARF19 directly regulate the auxin-mediated transcription of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16/ASL18) and/or LBD29/ASL16 in roots. Overexpression of LBD16/ASL18 and LBD29/ASL16 induces lateral root formation in the absence of ARF7 and ARF19. These LBD/ASL proteins are localized in the nucleus, and dominant repression of LBD16/ASL18 activity inhibits lateral root formation and auxin-mediated gene expression, strongly suggesting that these LBD/ASLs function downstream of ARF7-and ARF19-dependent auxin signaling in lateral root formation. Our results reveal that ARFs regulate lateral root formation via direct activation of LBD/ASLs in Arabidopsis.

Auxin regulates various aspects of plant growth and development. The AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode short-lived transcriptional repressors that are targeted by the TRANSPORT INHIBITOR RESPONSE1/AUXIN RECEPTOR F-BOX proteins. The Aux/IAA proteins regulate auxin-mediated gene expression by interacting with members of the AUXIN RESPONSE FACTOR protein family. Aux/IAA function is poorly understood; herein, we report the identification and characterization of insertion mutants in 12 of the 29 Aux/IAA family members. The mutants show no visible developmental defects compared with the wild type. Double or triple mutants of closely related Aux/IAA genes, such as iaa8-1 iaa9-1 or iaa5-1 iaa6-1 iaa19-1, also exhibit wild-type phenotypes. Global gene expression analysis reveals that the molecular phenotypes of auxin-treated and untreated light-grown seedlings are unaffected in the iaa17-6 and iaa5-1 iaa6-1 iaa19-1 mutants. By contrast, similar analysis with the gain-of-function axr3-1/iaa17-1 mutant seedlings reveals dramatic changes in basal and auxin-induced gene expression compared with the wild type. Expression of several type-A ARABIDOPSIS RESPONSE REGULATOR genes and a number of genes involved in cell wall biosynthesis and degradation is repressed in axr3-1/iaa17-1. The data suggest extensive functional redundancy among Aux/IAA gene family members and that enhanced stability of the AXR3/IAA17 protein severely alters the molecular phenotype, resulting in developmental defects.

SummaryAUXIN RESPONSE FACTORS (ARFs) regulate auxin-mediated transcriptional activation/repression. They are encoded by a gene family in Arabidopsis, and each member is thought to play a central role in various auxinmediated developmental processes. We have characterized three arf2 mutant alleles, arf2-6, arf2-7 and arf2-8. The mutants exhibit pleiotropic developmental phenotypes, including large, dark green rosette leaves, delayed flowering, thick and long inflorescence, abnormal flower morphology and sterility in early formed flowers, large organ size and delayed senescence and abscission, compared with wild-type plants. In addition, arf2 mutant seedlings have elongated hypocotyls with enlarged cotyledons under various light conditions. The transcription of ACS2, ACS6 and ACS8 genes is impaired in the developing siliques of arf2-6. The phenotypes of all three alleles are similar to those of the loss-of-function mutants obtained by RNA interference or co-suppression. There is no significant effect of the mutation on global auxin-regulated gene expression in young seedlings, suggesting that ARF2 does not participate in auxin signaling at that particular developmental stage of the plant life cycle. Because ARF2 is thought to function as a transcriptional repressor, the prospect arises that its pleiotropic effects may be mediated by negatively modulating the transcription of downstream genes in signaling pathways that are involved in cell growth and senescence.

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.root meristem | protein degradation D amaged DNA needs to be repaired to prevent loss or incorrect transmission of genetic information. Eukaryotic DNA damage checkpoints delay or arrest the cell cycle to provide time for DNA repair before the cell enters a new round of DNA replication or mitosis (1). In metazoans, ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that play a crucial role in the checkpoint system. ATM specifically responds to DNA double-strand breaks (DSBs), and ATR primarily senses replication stress caused by a persistent block of replication fork progression. ATM deficiency confers hypersensitivity to ionizing radiation (2), whereas ATR knockout mutation is lethal (3, 4), and dominant-negative cell lines display hypersensitivity to UVB light, gamma radiation, hydroxyurea (HU), and aphidicolin (5, 6). ATM and ATR relay the damage signal to transducer kinases Chk2 and Chk1, respectively, which then amplify the signal and regulate an overlapping set of substrates that trigger cell-cycle arrest and DNA repair (1). The transcription factor p53, cyclin-dependent kinase (CDK) inhibitor p21, and Cdc25 phosphatase are downstream regulators that control cell-cycle arrest in response to DNA damage.Comparative sequence analyses among plants, yeast, and animals indicate that some of the factors involved in DNA damage checkpoint and DSB repair systems are conserved between vertebrates and plants (7). Plants also possess ATM and ATR orthologs, and knockout mutants show similar phenotypes...

Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.

SummaryAuxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylemadjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/ indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.

In mammalian cells, the transcription factor p53 plays a crucial role in transmitting DNA damage signals to maintain genome integrity. However, in plants, orthologous genes for p53 and checkpoint proteins are absent. Instead, the plant-specific transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) controls most of the genes induced by gamma irradiation and promotes DNA repair, cell cycle arrest, and stem cell death. To date, the genes directly controlled by SOG1 remain largely unknown, limiting the understanding of DNA damage signaling in plants. Here, we conducted a microarray analysis and chromatin immunoprecipitation (ChIP)-sequencing, and identified 146 Arabidopsis genes as direct targets of SOG1. By using ChIP-sequencing data, we extracted the palindromic motif [CTT(N) AAG] as a consensus SOG1-binding sequence, which mediates target gene induction in response to DNA damage. Furthermore, DNA damage-triggered phosphorylation of SOG1 is required for efficient binding to the SOG1-binding sequence. Comparison between SOG1 and p53 target genes showed that both transcription factors control genes responsible for cell cycle regulation, such as CDK inhibitors, and DNA repair, whereas SOG1 preferentially targets genes involved in homologous recombination. We also found that defense-related genes were enriched in the SOG1 target genes. Consistent with this finding, SOG1 is required for resistance against the hemi-biotrophic fungus Colletotrichum higginsianum, suggesting that SOG1 has a unique function in controlling the immune response.