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.     

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.     

IAA17/AXR3: biochemical insight into an auxin mutant phenotype

The Aux/IAA genes are rapidly and specifically induced by the plant hormone auxin. The proteins encoded by this gene family are short-lived nuclear proteins that are capable of homodimerizing and heterodimerizing. Molecular, biochemical, and genetic data suggest that these proteins are involved in auxin signaling. The pleiotropic morphological phenotype and altered auxin responses of the semidominant axr3-1 mutant of Arabidopsis result from a single amino acid change in the conserved domain II of the Aux/IAA protein IAA17. Here, we show that the biochemical effect of this gain-of-function mutation is to increase the half-life of the iaa17/axr3-1 protein by sevenfold. Intragenic mutations that suppress the iaa17/axr3-1 phenotype have been described. The iaa17/axr3-1R3 revertant contains a second site mutation in domain I and the iaa17/axr3-1R2 revertant contains a second site mutation in domain III. Transient expression assays show that the mutant forms of IAA17/AXR3 retain the ability to accumulate in the nucleus. Using the yeast two hybrid system, we show that the iaa17/axr3-1 mutation does not affect homodimerization. However, the iaa17/axr3-1 revertants counteract the increased levels of iaa17/axr3-1 protein by decreasing the capacity of the mutant protein to homodimerize. Interestingly, heterodimerization of the revertant forms of IAA17/AXR3 with IAA3/SHY2, another Aux/IAA protein, and ARF1 or ARF5/MP proteins is affected only by changes in domain III. Collectively, the results provide biochemical evidence that the revertant mutations in the IAA17/AXR3 gene affect the capacity of the encoded protein to dimerize with itself, other members of the Aux/IAA protein family, and members of the ARF protein family. By extension, these findings may provide insight into the effects of analogous mutations in other members of the Aux/IAA gene family.     

Variation of dry matter accumulation at definite positions within wheat ears and levels of indole-3yl acetic acid (IAA)

By sterilization-treatments grain weights in defined positions within the wheat (Triticum aestivum L. cvs. Oskar, Kolibri, Norin 10) ear were varied. Main shoot ears were partitioned into basal, central and apical regions and further into proximal and distal grains. The central region was used for treatments and investigations. Proximal grains were sterilized in order to vary the dry weights of distal grains. Dry matter ac= cumulation and IAA levels were investigated during the grainfilling period. Results from two seasons are presented. In both seasons sterilization treatments accelerated the dry matter accumulation of distal grains in all cultivars, but treatment effects on IAA concentration depended upon interactions between years and cultivars. A parallel variation in grain weights and IAA concentrations occurred only in 1981 in the cultivar Oskar. In 1982, however, IAA concentrations were reduced in all cultivars. The results contradict an assumed causal correlation between dry matter storage and IAA concentration, but they do not exclude a possible role of IAA in regulating grain growth.     

Degradation of the auxin response factor ARF1

Auxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3–4 h. This half-life appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.     

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.     

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.