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.     

Teosinte Branched 1 modulates tillering in rice plants

Tillering is an important trait of cereal crops that optimizes plant architecture for maximum yield. Teosinte Branched 1 (TB1) is a negative regulator of lateral branching and an inducer of female inflorescence formation in Zea mays (maize). Recent studies indicate that TB1 homologs in Oryza sativa (rice), Sorghum bicolor and Arabidopsis thaliana act downstream of the auxin and MORE AUXILIARY GROWTH (MAX) pathways. However, the molecular mechanism by which rice produces tillers remains unknown. In this study, transgenic rice plants were produced that overexpress the maize TB1 (mTB1) or rice TB1 (OsTB1) genes and silence the OsTB1 gene through RNAi-mediated knockdown. Because lateral branching in rice is affected by the environmental conditions, the phenotypes of transgenic plants were observed in both the greenhouse and the paddy field. Compared to wild-type plants, the number of tillers and panicles was reduced and increased in overexpressed and RNAi-mediated knockdown OsTB1 rice plants, respectively, under both environmental conditions. However, the effect was small for plants grown in paddy fields. These results demonstrate that both mTB1 and OsTB1 moderately regulate the tiller development in rice.     

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.     

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

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.     

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.     

OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.)

Members of the ATP Binding Cassette B/Multidrug‐Resistance/P–glyco‐protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock‐down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4‐dichlorophenoxyacetic acid (2,4–D) and indole‐3‐acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock‐down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (–Fe). Their Fe concentration is higher and upregulation of Fe deficiency‐responsive genes is lower in osabcb14 mutants than in wild‐type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition.     

Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid‐mediated defenses

Auxin is a key plant growth regulator that also impacts plant–pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole‐3‐acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector‐triggered immunity was active in YUC1‐overexpressing plants, and we observed only minor effects on SA levels and SA‐mediated responses. Furthermore, a plant line carrying both the YUC1‐overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA‐mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA‐mediated defenses.     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.