Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

The analysis of auxin distribution in the wild type and <Emphasis Type="Italic">abruptus</Emphasis> mutant plants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) heynh. By using the chimeric gene <Emphasis Type="Italic">DR5::GUS</Emphasis>

We have studied the activity of the chimeric gene DR5::GUS that indicates the spatial pattern of free auxin distribution and relative auxin level in the tissues of A. thaliana plants. It is established that the temperature sensitive abruptus mutation leads to temperature dependent increased accumulation of the free auxin in leaves, staments, and sepals in comparison to control plants homozygous for the wild type allele ABRUPTUS/PINOID. Our data revealed the important role of the ABRUPTUS/PINOID gene in the regulation of the auxin polar transport.     

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.     

Interactions between the ABRUPTUS/PINOID and LEAFY Genes during Floral Morphogenesis in Arabidopsis thaliana (L.) Heynh.

Analysis of interaction between mutations abruptus andleafy and previous data on interactions of abruptuswith homeotic mutations apetala1, apetala2, and apetala3 showed that the functions of the ABRUPTUS/PINOID (ABR/PID) gene are as follows: (1) it determines position of lateral organs on the inflorescence without specifying their identity [floral meristem (FM) or cauline leaves]; (2) in concert with theLEAFY (LFY) gene, it participates in the formation of FM; (3) it is involved in the determination and the formation of floral organ primordia in the first, second, and third whorls. Auxin accumulation in the abr mutant cells in callus culture was shown indicating the involvement of the ABR/PID gene in regulation of auxin efflux from cells. It is suggested that the ABR/PID expression in the sites of formation of FM and floral organs leads to local reduction in auxin level and/or activation of the lateral auxin flow, which in turn, enhance expression of the LFYand homeotic genes responsible for FM formation and differentiation.     

Growth and Development in Arabidopsis thaliana through an Entire Life Cycle under Simulated Microgravity Conditions on a Clinostat

Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin-formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes. Received 18 June 1999/ Accepted in revised form 27 August 1999     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development

MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.     

The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Arabidopsis pinoid mutants show a strong phenotypic resemblance to the pin-formed mutant that is disrupted in polar auxin transport. The PINOID gene was recently cloned and found to encode a protein-serine/threonine kinase. Here we show that the PINOID gene is inducible by auxin and that the protein kinase is present in the primordia of cotyledons, leaves and floral organs and in vascular tissue in developing organs or proximal to meristems. Overexpression of PINOID under the control of the constitutive CaMV 35S promoter (35S::PID) resulted in phenotypes also observed in mutants with altered sensitivity to or transport of auxin. A remarkable characteristic of high expressing 35S::PID seedlings was a frequent collapse of the primary root meristem. This event triggered lateral root formation, a process that was initially inhibited in these seedlings. Both meristem organisation and growth of the primary root were rescued when seedlings were grown in the presence of polar auxin transport inhibitors, such as naphthylphtalamic acid (NPA). Moreover, ectopic expression of PINOID cDNA under control of the epidermis-specific LTP1 promoter provided further evidence for the NPA-sensitive action of PINOID. The results presented here indicate that PINOID functions as a positive regulator of polar auxin transport. We propose that PINOID is involved in the fine-tuning of polar auxin transport during organ formation in response to local auxin concentrations.     

Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation

The pin-formed mutant pin 1-1, one of the Arabidopsis flower mutants, has several structural abnormalities in inflorescence axes, flowers, and leaves. In some cases, pin1-1 forms a flower with abnormal structure (wide petals, no stamens, pistil-like structure with no ovules in the ovary) at the top of inflorescence axes. In other cases, no floral buds are formed on the axes. An independently isolated allelic mutant (pin1-2) shows similar phenotypes. These mutant phenotypes are exactly the same in wild-type plants cultured in the presence of chemical compounds known as auxin polar transport inhibitors: 9-hydroxyfluorene-9-carboxylic acid or N-(1-naphthyl)phthalamic acid. We tested the polar transport activity of indole-3-acetic acid and the endogenous amount of free indole-3-acetic acid in the tissue of inflorescence axes of the pin1 mutants and wild type. The polar transport activity in the pin 1-1 mutant and in the pin1-2 mutant was decreased to 14% and 7% of wild type, respectively. These observations strongly suggest that the normal level of polar transport activity in the inflorescence axes is required in early developmental stages of floral bud formation in Arabidopsis and that the primary function of the pin1 gene is auxin polar transport in the inflorescence axis.     

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.     

Interplay between the NADP-Linked Thioredoxin and Glutathione Systems in Arabidopsis Auxin Signaling

Intracellular redox status is a critical parameter determining plant development in response to biotic and abiotic stress. Thioredoxin (TRX) and glutathione are key regulators of redox homeostasis, and the TRX and glutathione pathways are essential for postembryonic meristematic activities. Here, we show by associating TRX reductases (ntra ntrb) and glutathione biosynthesis (cad2) mutations that these two thiol reduction pathways interfere with developmental processes through modulation of auxin signaling. The triple ntra ntrb cad2 mutant develops normally at the rosette stage, undergoes the floral transition, but produces almost naked stems, reminiscent of the phenotype of several mutants affected in auxin transport or biosynthesis. In addition, the ntra ntrb cad2 mutant shows a loss of apical dominance, vasculature defects, and reduced secondary root production, several phenotypes tightly regulated by auxin. We further show that auxin transport capacities and auxin levels are perturbed in the mutant, suggesting that the NTR-glutathione pathways alter both auxin transport and metabolism. Analysis of ntr and glutathione biosynthesis mutants suggests that glutathione homeostasis plays a major role in auxin transport as both NTR and glutathione pathways are involved in auxin homeostasis.     

Genetic regulation of vascular tissue patterning in Arabidopsis

Plants transport water and nutrients through a complex vascular network comprised of interconnected, specialized cell types organized in discrete bundles. To identify genetic determinants of vascular tissue patterning, we conducted a screen for mutants with altered vascular bundle organization in Arabidopsis cotyledons. Mutations in two genes, CVP1 and CVP2 (for cotyledon vascular pattern), specifically disrupt the normal pattern of vascular bundles in cotyledons, mature leaves, and inflorescence stems. The spatial distribution of the procambium, the precursor to mature vascular tissue, is altered in cvp1 and cvp2 embryos, suggesting that CVP1 and CVP2 act at a very early step in vascular patterning. Similarly, in developing stems of cvp1 and leaves of cvp2, the pattern of vascular differentiation is defective, but the maturation of individual vascular cells appears to be normal. There are no discernible alterations in cell morphology in cvp2 mutants. In contrast, cvp1 mutants are defective in directional orientation of the provascular strand, resulting in a failure to establish uniformly aligned vascular cells, and they also show a reduction in vascular cell elongation. Neither cvp1 nor cvp2 mutants displayed altered auxin perception, biosynthesis, or transport, suggesting that auxin metabolism is not generally affected in these mutants.     

ABC transporters coordinately expressed during lignification of Arabidopsis stems include a set of ABCBs associated with auxin transport

The primary inflorescence stem of Arabidopsis thaliana is rich in lignified cell walls, in both vascular bundles and interfascicular fibres. Previous gene expression studies demonstrated a correlation between expression of phenylpropanoid biosynthetic genes and a subset of genes encoding ATP-binding cassette (ABC) transporters, especially in the ABCB/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) and ABCG/pleiotropic drug resistance (ABCG/PDR) subfamilies. The objective of this study was to characterize these ABC transporters in terms of their gene expression and their function in development of lignified cells. Based on in silico analyses, four ABC transporters were selected for detailed investigation: ABCB11/MDR8, ABCB14/MDR12, ABCB15/MDR13, and ABCG33/PDR5. Promoter::glucuronidase reporter assays for each gene indicated that promoters of ABCB11, ABCB14, ABCB15, and ABCG33 transporters are active in the vascular tissues of primary stem, and in some cases in interfascicular tissues as well. Homozygous T-DNA insertion mutant lines showed no apparent irregular xylem phenotype or alterations in interfascicular fibre lignification or morphology in comparison with wild type. However, in abcb14-1 mutants, stem vascular morphology was slightly disorganized, with decreased phloem area in the vascular bundle and decreased xylem vessel lumen diameter. In addition, abcb14-1 mutants showed both decreased polar auxin transport through whole stems and altered auxin distribution in the procambium. It is proposed that both ABCB14 and ABCB15 promote auxin transport since inflorescence stems in both mutants showed a reduction in polar auxin transport, which was not observed for any of the ABCG subfamily mutants tested. In the case of ABCB14, the reduction in auxin transport is correlated with a mild disruption of vascular development in the inflorescence stem.     

SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis

The regular arrangement of leaves and flowers around a plant''s stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.     

Auxin Polar Transport Is Essential for the Establishment of Bilateral Symmetry during Early Plant Embryogenesis

We used an in vitro culture system to investigate the effects of three auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, trans-cinnamic acid, and 2,3,5-triiodobenzoic acid) on the development of early globular to heart-shaped stage embryos of Indian mustard (Brassica juncea) plants. Although the effective concentrations vary with the different inhibitors, all of them induced the formation of fused cotyledons in globular ([less than or equal to]60 [mu]m) but not heart-shaped embryos. Inhibitor-treated Brassica embryos phenocopy embryos of the Arabidopsis pin-formed mutant pin1-1, which has reduced auxin polar transport activity in inflorescence axes, as well as embryos of the Arabidopsis emb30 (gnom) mutant. These results indicate that the polar transport of auxin in early globular embryos is essential for the establishment of bilateral symmetry during plant embryogenesis. Based on these observations, we propose two possible models for the action of auxin during cotyledon formation.     

Redox regulation of auxin signaling and plant development in Arabidopsis

Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems that play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,¹ we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.Key words: arabidopsis, NTS pathway, NGS pathway, thioredoxin (TRX), glutaredoxine (GRX), polar auxin transport (PAT), auxin biosynthesis, pin-like phenotype, apical dominance, meristematic activityExposure of living organisms to environmental stresses triggers various defense and developmental responses. Redox signaling is involved in many aspects of these responses.^2–6 The key players in these responses are the NADPH-dependent glutathione/glutaredoxin system (NGS) and the NADPH-dependent thioredoxin system (NTS). TRX and GRX play key roles in the maintenance of cellular redox homeostasis.^7–10 Genetic approaches aiming to identify functions of TRX and GRX in knock-out plants have largely been limited by the absence of phenotypes of single mutants, presumably due to functional redundancies among members of the multigene families of TRX and GRX.¹¹ Interplay between NTS and NGS pathways have been studied in different organisms^12–17 and association of mutants involved in these two pathways have recently revealed new functions in several aspects of plant development.^4–6     

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5_rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

mtHSC70-1 dysfunction induces mitochondrial retrograde regulation, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.