Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana.

Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI.

Lateral Inhibition by a Peptide Hormone-Receptor Cascade during Arabidopsis Lateral Root Founder Cell Formation.

In plants, the position of lateral roots (LRs) depends on initiation sites induced by auxin. The domain of high auxin response responsible for LR initiation stretches over several cells, but only a pair of pericycle cells (LR founder cells) will develop into LRs. In this work, we identified a signaling cascade controlling LR formation through lateral inhibition. It comprises a peptide hormone TARGET OF LBD SIXTEEN 2 (TOLS2), its receptor RLK7, and a downstream transcription factor PUCHI. TOLS2 is expressed at the LR founder cells and inhibits LR initiation. Time-lapse imaging of auxin-responsive DR5:LUCIFERASE reporter expression revealed that occasionally two pairs of LR founder cells are specified in close proximity even in wild-type and that one of them exists only transiently and disappears in an RLK7-dependent manner. We propose that the selection of LR founder cells by the peptide hormone-receptor cascade ensures proper LR spacing.

Identifying the target genes of SUPPRESSOR OF GAMMA RESPONSE 1, a master transcription factor controlling DNA damage response in Arabidopsis.

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)7 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.

Differential regulation of B2-type CDK accumulation in Arabidopsis roots.

KEY MESSAGE: The accumulation of the mitotic B2-type CDK is tightly controlled by multiple pathways in Arabidopsis roots. Root growth depends on cell proliferation in the apices, which determines the root meristem size. The expression of B2-type cyclin-dependent kinase (CDKB2) is known to be restricted to dividing cells in the meristematic region, and therefore, the mechanisms controlling CDKB2 accumulation may be associated with those determining the meristem size. We investigated how CDKB2 expression is controlled in distinct zones of Arabidopsis roots. We found that CDKB2;1 expression was induced by a member of the PLETHORA (PLT) family of transcription factors, which are known to mediate auxin signaling and maintain the undifferentiated state of meristematic cells. When the root meristem was treated with an auxin antagonist, the CDKB2;1 level was reduced not only by transcriptional suppression but also by proteasome-mediated protein degradation. This indicates that auxin promotes CDKB2 accumulation at both mRNA and protein levels in the meristem. In the elongation and differentiation zones, on the other hand, neither the ubiquitin-proteasome system nor the PLT-mediated transcriptional regulation is associated with CDKB2;1 accumulation. Both CDKB2;1 and HIGH PLOIDY2 (HPY2), a SUMO E3 ligase, were ectopically accumulated in the stele when treated with exogenous auxin, suggesting the possibility that CDKB2;1 accumulation is dependent on HPY2-mediated sumoylation, which is usually maintained by a higher auxin level in the meristem. Our results demonstrate that the CDKB2 level is tightly controlled by multiple pathways to maintain the mitotic activity in developing roots.

The Arabidopsis SIAMESE-RELATED cyclin-dependent kinase inhibitors SMR5 and SMR7 regulate the DNA damage checkpoint in response to reactive oxygen species.

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.

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.

Restriction of cell proliferation in internal tissues via the synthesis of very-long-chain fatty acids in the epidermis.

Very-long-chain fatty acids (VLCFAs) are major components of cuticular wax and are also contained in seed storage triacylglycerols and sphingolipids. Arabidopsis mutants with severe defects in VLCFA synthesis produce fused leaves because of impaired cuticle formation. We recently reported that a small decrease in VLCFA content did not cause growth defects, but instead led to enhanced cell proliferation in internal tissues. We observed that this overproliferation was induced by elevated expression of cytokinin biosynthesis genes, which in turn increased the cytokinin level. Interestingly, VLCFAs are specifically synthesized in the epidermis for cuticular wax secretion, whereas cytokinin biosynthesis mainly occurs in the vasculature. Our results indicate the requirement of VLCFA synthesis in the epidermis for sending non-autonomous signals, thereby suppressing cytokinin biosynthesis in the vasculature. We propose that the interaction between the surface (epidermis) and axis (vasculature) of the plant body fine-tunes cell division activity and restricts organ size in determinate growth.

Synthesis of very-long-chain fatty acids in the epidermis controls plant organ growth by restricting cell proliferation.

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.

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.

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.

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.

ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes 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.

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.

Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis.

Auxin 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 protoxylem-adjacent 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.

AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator.

AUXIN 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 auxin-mediated 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.

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.

Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa.

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.