Yoko Okushima

Functional Genomic Analysis of the AUXIN RESPONSE FACTOR Gene Family Members in Arabidopsis thaliana: Unique and Overlapping Functions of ARF7 and ARF19

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.

Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana

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.

Auxin-mediated lateral root formation in higher plants.

Lateral root (LR) formation is an important organogenetic process that contributes to the establishment of root architecture in higher plants. In the angiosperms, LRs are initiated from the pericycle, an inner cell layer of the parent roots. Auxin is a key plant hormone that promotes LR formation, but the molecular mechanisms of auxin-mediated LR formation remain unknown. Molecular genetic studies using Arabidopsis mutants have revealed that the auxin transport system with a balance of influx and efflux is important for LR initiation and subsequent LR primordium development. In addition, normal auxin signaling mediated by two families of transcriptional regulators, Aux/IAAs and ARFs, is necessary for LR formation. This article is an update on the mechanisms of auxin-mediated LR formation in higher plants, particularly in Arabidopsis.

Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis

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.

Domain II Mutations in CRANE/IAA18 Suppress Lateral Root Formation and Affect Shoot Development in Arabidopsis thaliana

Lateral root formation is an important developmental component of root systems in vascular plants. Several regulatory genes for lateral root formation have been identified from recent studies mainly using Arabidopsis thaliana. In this study, we isolated two dominant mutant alleles, crane-1 and crane-2, which are defective in lateral root formation in Arabidopsis. The crane mutants have dramatically reduced lateral root and auxin-induced lateral root formation, indicating that the crane mutations reduce auxin sensitivity. In addition, the crane mutants have pleiotropic phenotypes in the aerial shoots, including long hypocotyls when grown in the light, up-curled leaves and reduced fertility. The crane mutant phenotypes are caused by a gain-of-function mutation in domain II of IAA18, a member of the Aux/IAA transcriptional repressor family which is expressed in almost all organs. In roots, IAA18 promoter::GUS was expressed in the early stages of lateral root development. In the yeast two-hybrid system, IAA18 interacts with AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19, transcriptional activators that positively regulate lateral root formation. Taken together, our results indicate that CRANE/IAA18 is involved in lateral root formation in Arabidopsis, and suggest that it negatively regulates the activity of ARF7 and ARF19 for lateral root formation.

The Arabidopsis SIAMESE-RELATED Cyclin-Dependent Kinase Inhibitors SMR5 and SMR7 Regulate the DNA Damage Checkpoint in Response to Reactive Oxygen Species

Reactive oxygen species (ROS) cause DNA damage. In this work, two SIAMESE/SIAMESE-RELATED (SIM/SMR) genes that encode cyclin-dependent kinase inhibitors are described as being part of a signaling pathway that arrests cell proliferation in response to ROS, revealing a novel cell cycle checkpoint-signaling cascade. 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.

Synthesis of Very-Long-Chain Fatty Acids in the Epidermis Controls Plant Organ Growth by Restricting Cell Proliferation

The synthesis of very-long-chain fatty acids (VLCFAs) in the epidermis is essential for the proper control of cell growth in Arabidopsis. VLCFAs act via their ability to suppress cytokinin synthesis in the vasculature, thus preventing cell overproliferation in internal tissues.

Cytokinins Control Endocycle Onset by Promoting the Expression of an APC/C Activator in Arabidopsis Roots

Plant roots respond to various internal and external signals and adjust themselves to changes of environmental conditions. In the root meristem, stem cells produce daughter cells that continue to divide several times. When these latter cells reach the transition zone, they stop dividing and enter the endocycle, a modified cell cycle in which DNA replication is repeated without mitosis or cytokinesis. The resultant DNA polyploidization, named endoreduplication, is usually associated with an increase of nuclear and cell volume and with cell differentiation. At the transition zone, cytokinin signaling activates two transcription factors, type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12, and induces SHY2/IAA3, a member of the Aux/IAA family of auxin signaling repressors. This inhibits auxin signaling and reduces the expression of auxin efflux carriers, resulting in cell division arrest. Such counteracting actions of two hormones are assumed to determine meristem size. However, it remains unknown whether cytokinins additionally control meristem size through an auxin-independent pathway. Here we show that, in Arabidopsis, the cytokinin-activated ARR2 directly upregulates the expression of CCS52A1, which encodes an activator of an E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C), thereby promoting the onset of the endocycle and restricting meristem size. Our genetic data revealed that CCS52A1 function is independent of SHY2-mediated control of auxin signaling, indicating that downregulation of auxin signaling and APC/C-mediated degradation of cell-cycle regulators cooperatively promote endocycle onset, and thus fine tune root growth.

Kip-Related Protein 3 Is Required for Control of Endoreduplication in the Shoot Apical Meristem and Leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.

Glycosylation and its Adequate Processing is Critical for Protein Secretion in Tobacco BY2 Cells

Summary Peroxidases are secreted into the culture medium of BY2 suspension cells. In order to determine the role of glycosylation and glycan processing for peroxidase secretion, inhibition studies were performed. When glycosylation was inhibited by tunicamycin, secretion of peroxidases into the medium ceased totally. When glycan processing was inhibited by castanospermine, the molecular size of the peroxidases was increased concomitant with their decreased secretion into the medium. Upon inhibition treatment, pulse-labeled total secreted protein behaved similarly to the peroxidases, indicating that for secretion, glycosylation is generally essential, while glycan processing is not prerequisite but none the less necessary. These studies are supported by the observation that inhibitor treatment also induced transcript accumulation for the molecular chaperon, BiP, and for calnexin, which recognizes immature glycans. Taken together, these results strongly suggest that correct N-glycan structure is critical for protein secretion in plant cells.