The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin-regulated gene SAUR-AC1 in mutant and wild-type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin-induced accumulation of SAUR-AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR-AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an inter-action between the AXR1 and AUX1 genes. In mature plants, the aux1-7 mutation acts to partially suppress the morphological defects conferred by the axr1-12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR-AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.     

Expression pattern of Aux/IAA genes in the iaa3/shy2-1D mutant of Arabidopsis thaliana (L.)

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.     

WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.     

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.     

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.     

磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

The blue light receptor Phototropin 1 suppresses lateral root growth by controlling cell elongation

We investigated the relationship between the blue light receptor phototropin 1 (phot1) and lateral root growth in Arabidopsis thaliana seedlings. Fluorescence and confocal microscopy images, as well as PHOT1 mRNA expression studies provide evidence that it is highly expressed in the elongation zone of lateral roots where auxin is accumulating. However, treatment with the auxin transport inhibitor N‐1‐naphthylphthalamic acid significantly reduced PHOT1 expression in this zone. In addition, PHOT1 expression was higher in darkness than in light. The total number of lateral roots was higher in the phot1 mutant than in wild‐type Arabidopsis. Cells in the elongation zone of lateral roots of the phot1 mutant were longer than those of wild‐type lateral roots. These findings suggest that PHOT1 plays a role(s) in elongation of lateral roots through the control of an auxin‐related signalling pathway.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (x 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR-related SAX1 gene.     

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N -1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.     

Maize VP1 complements Arabidopsisabi3 and confers a novel ABA/auxin interaction in roots

The maize Vp1 gene and abi3 gene of Arabidopsis are believed to be orthologs based on similarities of the mutant phenotypes and amino acid sequence conservation. Here we show that expression of VP1 driven by the 35S promoter can partially complement abi3-6, a deletion mutant allele of abi3. The visible phenotype of seed produced from VP1 expression in the abi3 mutant background is nearly indistinguishable from wild type. VP1 fully restores abscisic acid (ABA) sensitivity of abi3 during seed germination and suppresses the early flowering phenotype of abi3. The temporal regulation of C1-beta-glucuronidase (GUS) and chlorophyll a/b binding protein (cab3)-GUS reporter genes in developing seeds of 35S-VP1 lines were similar to wild type. On the other hand, two qualitative differences are observed between the 35S-VP1 line and wild type. The levels of CRC and C1-GUS expression are markedly lower in the seeds of 35S-VP1 lines than in wild type suggesting incomplete complementation of gene activation functions. Similar to ectopic expression of ABI3 (Parcy et al., 1994), ectopic expression of VP1 in vegetative tissue enhances ABA inhibition of root growth. In addition, 35S-VP1 confers strong ABA inducible expression of the normally seed-specific cruciferin C (CRC) gene in leaves. In contrast, ectopic ABA induction of C1-GUS is restricted to a localized region of the root elongation zone. The ABA-dependent C1-GUS expression expanded to a broader area in the root tissues treated with exogenous application of auxin. Interestingly, auxin-induced lateral root formation is completely suppressed by ABA in 35S-VP1 plants but not in wild type. These results indicate VP1 mediates a novel interaction between ABA and auxin signaling that results in developmental arrest and altered patterns of gene expression.     

Growth and development of the axr1 mutants of Arabidopsis.

We have recovered eight new auxin-resistant lines of Arabidopsis that carry mutations in the AXR1 gene. These eight lines, together with the 12 lines described in a previous report, define at least five different axr1 alleles. All of the mutant lines have a similar phenotype. Defects include decreases in plant height, root gravitropism, hypocotyl elongation, and fertility. Mutant line axr1-3 is less resistant to auxin than the other mutant lines and has less severe morphological abnormalities. This correlation suggests that the morphological defects are a consequence of a defect in auxin action. To determine whether the altered morphology of mutant plants is associated with changes in cell size or tissue organization, tissue sections were examined using scanning electron microscopy. No clear differences in cell size were observed between wild-type and mutant tissues. However, the vascular bundles of mutant stems were found to be less well differentiated than those in wild-type stems. The auxin sensitivity of rosette-stage plants was determined by spraying plants with auxin solutions. Mutant rosettes were found to be significantly less sensitive to exogenously applied auxin than wild-type rosettes, indicating that the AXR1 gene functions in aerial portions of the plant. Our studies suggest that the AXR1 gene is required for auxin action in most, if not all, tissues of the plant and plays an important role in plant development. Linkage studies indicate that the gene is located on chromosome 1 approximately 2 centiMorgans from the closest restriction fragment length polymorphism.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.