Multiple AUX/IAA�CARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling

In Arabidopsis thaliana, lateral root (LR) formation is regulated by multiple auxin/indole-3-acetic acid (Aux/IAA)–AUXIN RESPONSE FACTOR (ARF) modules: (i) the IAA28–ARFs module regulates LR founder cell specification; (ii) the SOLITARY-ROOT (SLR)/IAA14–ARF7–ARF19 module regulates nuclear migration and asymmetric cell divisions of the LR founder cells for LR initiation; and (iii) the BODENLOS/IAA12–MONOPTEROS/ARF5 module also regulates LR initiation and organogenesis. The number of Aux/IAA–ARF modules involved in LR formation remains unknown. In this study, we isolated the shy2-101 mutant, a gain-of-function allele of short hypocotyl2/suppressor of hy2 (shy2)/iaa3 in the Columbia accession. We demonstrated that the shy2-101 mutation not only strongly inhibits LR primordium development and emergence but also significantly increases the number of LR initiation sites with the activation of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18, a target gene of the SLR/IAA14–ARF7–ARF19 module. Genetic analysis revealed that enhanced LR initiation in shy2-101 depended on the SLR/IAA14–ARF7–ARF19 module. We also showed that the shy2 roots contain higher levels of endogenous IAA. These observations indicate that the SHY2/IAA3–ARF-signalling module regulates not only LR primordium development and emergence after SLR/IAA14–ARF7–ARF19 module-dependent LR initiation but also inhibits LR initiation by affecting auxin homeostasis, suggesting that multiple Aux/IAA–ARF modules cooperatively regulate the developmental steps during LR formation.     

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 signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis

Auxin signaling mediated by various auxin/indole‐3‐acetic acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of the SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin‐inducible gene. The precise developmental role and molecular regulation of LRP1 in root development remain to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides the primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin‐dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs‐ARFs modules during LR development. We showed that auxin‐mediated induction of LRP1 is lost in emerging LRs of slr‐1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric division during LR development. Overexpression of LRP1 (LRP1 OE) showed an increased number of LR primordia (LRP) at stages I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1‐induced expression of YUC4, which is involved in auxin biosynthesis, contributes to the increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3, SRS6 and SRS7 proteins of the SHI/STY family, indicating their possible redundant role during root development. Our results suggested that auxin and histone deacetylation affect LRP1 expression and it acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.     

Multiple AUX/IAA-ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling

In Arabidopsis thaliana, lateral root (LR) formation is regulated by multiple auxin/indole-3-acetic acid (Aux/IAA)-AUXIN RESPONSE FACTOR (ARF) modules: (i) the IAA28-ARFs module regulates LR founder cell specification; (ii) the SOLITARY-ROOT (SLR)/IAA14-ARF7-ARF19 module regulates nuclear migration and asymmetric cell divisions of the LR founder cells for LR initiation; and (iii) the BODENLOS/IAA12-MONOPTEROS/ARF5 module also regulates LR initiation and organogenesis. The number of Aux/IAA-ARF modules involved in LR formation remains unknown. In this study, we isolated the shy2-101 mutant, a gain-of-function allele of short hypocotyl2/suppressor of hy2 (shy2)/iaa3 in the Columbia accession. We demonstrated that the shy2-101 mutation not only strongly inhibits LR primordium development and emergence but also significantly increases the number of LR initiation sites with the activation of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18, a target gene of the SLR/IAA14-ARF7-ARF19 module. Genetic analysis revealed that enhanced LR initiation in shy2-101 depended on the SLR/IAA14-ARF7-ARF19 module. We also showed that the shy2 roots contain higher levels of endogenous IAA. These observations indicate that the SHY2/IAA3-ARF-signalling module regulates not only LR primordium development and emergence after SLR/IAA14-ARF7-ARF19 module-dependent LR initiation but also inhibits LR initiation by affecting auxin homeostasis, suggesting that multiple Aux/IAA-ARF modules cooperatively regulate the developmental steps during LR formation.     

Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice ( Oryza sativa ), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring m OsIAA3 - GR , which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that , in rice, auxin is important for development and its signaling is mediated by IAA genes.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

Auxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down-regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling-mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh-mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.     

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein,Enhancing Viral Infection and Disease Development

The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.     

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins,plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin‐responsive [auxin (AUX)/indole‐3‐acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF^TIR to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin‐related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two‐hybrid and in vitro pull‐down results revealed an association between OsCYP2 and the co‐chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1‐naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.