Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.     

Responses of plant vascular systems to auxin transport inhibition.

To assess the role of auxin flows in plant vascular patterning, the development of vascular systems under conditions of inhibited auxin transport was analyzed. In Arabidopsis, nearly identical responses evoked by three auxin transport inhibitor substances revealed an enormous plasticity of the vascular pattern and suggest an involvement of auxin flows in determining the sites of vascular differentiation and in promoting vascular tissue continuity. Organs formed under conditions of reduced auxin transport contained increased numbers of vascular strands and cells within those strands were improperly aligned. In leaves, vascular tissues became progressively confined towards the leaf margin as the concentration of auxin transport inhibitor was increased, suggesting that the leaf vascular system depends on inductive signals from the margin of the leaf. Staged application of auxin transport inhibitor demonstrated that primary, secondary and tertiary veins became unresponsive to further modulations of auxin transport at successive stages of early leaf development. Correlation of these stages to anatomical features in early leaf primordia indicated that the pattern of primary and secondary strands becomes fixed at the onset of lamina expansion. Similar alterations in the leaf vascular responses of alyssum, snapdragon and tobacco plants suggest common functions of auxin flows in vascular patterning in dicots, while two types of vascular pattern alterations in Arabidopsis auxin transport mutants suggest that at least two distinct primary defects can result in impaired auxin flow. We discuss these observations with regard to the relative contributions of auxin transport, auxin sensitivity and the cellular organisation of the developing organ on the vascular pattern.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

Developmental role and auxin responsiveness of Class III homeodomain leucine zipper gene family members in rice

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner.     

Auxin transport promotes Arabidopsis lateral root initiation

Lateral root development in Arabidopsis provides a model for the study of hormonal signals that regulate postembryonic organogenesis in higher plants. Lateral roots originate from pairs of pericycle cells, in several cell files positioned opposite the xylem pole, that initiate a series of asymmetric, transverse divisions. The auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) arrests lateral root development by blocking the first transverse division(s). We investigated the basis of NPA action by using a cell-specific reporter to demonstrate that xylem pole pericycle cells retain their identity in the presence of the auxin transport inhibitor. However, NPA causes indoleacetic acid (IAA) to accumulate in the root apex while reducing levels in basal tissues critical for lateral root initiation. This pattern of IAA redistribution is consistent with NPA blocking basipetal IAA movement from the root tip. Characterization of lateral root development in the shoot meristemless1 mutant demonstrates that root basipetal and leaf acropetal auxin transport activities are required during the initiation and emergence phases, respectively, of lateral root development.     

Inhibition of Auxin Movement from the Shoot into the Root Inhibits Lateral Root Development in Arabidopsis

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [³H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.     

Putative dual pathway of auxin transport in organogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, damage to the superficial acropetal polar auxin transport (PAT) inhibits generative but not vegetative organ initiation. In order to verify whether in a vegetative phase auxin can be transported to the meristem in a different way, the research on wild-type and plants with defective PAT was performed. Distance from the differentiated vascular elements to the shoot apical meristem (SAM) was measured for Arabidopsis cultured in different experimental systems. The influence of this distance on the ability to induce organogenesis as well as transport of the fluorescent dye to the SAM, and the LEAFY gene expression were analyzed. The youngest protoxylem elements were used as a marker of the vascular tissues. The distance of protoxylem to the SAM and organogenesis were interrelated. Organ initiation occurred only when protoxylem was localized near to the SAM. Experimental elongation of internodes in a vegetative rosette caused an increase in the distance between protoxylem and the SAM organogenic zone. Thus, the inhibition of organ initiation took place already during the vegetative phase. The results suggest the presence of at least two pathways of acropetal transport of auxin inducing organogenesis: one superficial way through PAT, and the second, putative one, internal through the vascular system. Possibly, organogenesis is completely blocked only when both these pathways are dysfunctional.     

Model for the role of auxin polar transport in patterning of the leaf adaxial–abaxial axis

Leaf adaxial–abaxial polarity refers to the two leaf faces, which have different types of cells performing distinct biological functions. In 1951, Ian Sussex reported that when an incipient leaf primordium was surgically isolated by an incision across the vegetative shoot apical meristem (SAM), a radialized structure without an adaxial domain would form. This led to the proposal that a signal, now called the Sussex signal, is transported from the SAM to emerging primordia to direct leaf adaxial–abaxial patterning. It was recently proposed that instead of the Sussex signal, polar transport of the plant hormone auxin is critical in leaf polarity formation. However, how auxin polar transport functions in the process is unknown. Through live imaging, we established a profile of auxin polar transport in and around young leaf primordia. Here we show that auxin polar transport in lateral regions of an incipient primordium forms auxin convergence points. We demonstrated that blocking auxin polar transport in the lateral regions of the incipient primordium by incisions abolished the auxin convergence points and caused abaxialized leaves to form. The lateral incisions also blocked the formation of leaf middle domain and margins and disrupted expression of the middle domain/margin‐associated marker gene WUSCHEL‐RELATED HOMEOBOX 1 (SlWOX1). Based on these results we propose that the auxin convergence points are required for the formation of leaf middle domain and margins, and the functional middle domain and margins ensure leaf adaxial–abaxial polarity. How middle domain and margins function in the process is discussed.     

Glucose-6-phosphate-dehydrogenase activity in the shoot apical meristem,leaf primordia,and leaf tissues of Dianthus chinensis L.

The oxidation of carbohydrate by the pentose-phosphate pathway in the shoot apical meristem and developing leaf primordia of Dianthus chinensis was assessed by measuring the activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). On a kg^-1 dry weight h^-1 basis, activity rose from 250 mmol in the apical meristem to 550 mmol in the first two leaf primordia and then declined to 350 mmol in the sixth pair of leaf primordia, and finally to 200 mmol in leaves just emerged from the shoot bud. Measurements of activity in the sixth leaf pair from the apex showed differential distribution in leaf tissues. Epidermal and mesophyll tissue had about the same activity as whole-leaf tissue, but vascular bundles had 70% greater activity. Within the vascular tissue, activity in the phloem was twice as high as in the xylem. When activity was expressed on a per-cell basis, there was a continuous increase from 20 fmol in the apex to 2 pmol in the sixth leaf pair. Activity on a per unit cell volume basis showed that cells of the apical meristem and the epidermis, mesophyll and xylem of the sixth leaf pair had similar values, about 30 amol; only the two youngest pairs of primordia and the phloem had values two or three times this amount.     

DEVELOPMENTAL POTENTIAL OF EXCISED PRIMORDIAL AND EXPANDING LEAVES OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. primordial leaves 1 through 4 and expanding leaves 5 to 8 were isolated and cultured to examine the effects of auxin and kinetin on development. Without the plant growth regulators in the medium, expanding leaves 7 and 8 developed as leaves; younger leaf primordia did not develop. With 0.01 to 5.0 mg/1 IAA, 2–7% of the youngest pair of primordial leaves (1 and 2) developed as roots. Small leaf blade development occurred on IAA at 0.5 to 5.0 mg/1 with 10–12% of the explants, and shoots developed from 2% of the youngest primordia explants at 3 mg/1 IAA. With 2–28% of the second set of primordial leaves (3 and 4), a leaf with a root developed with 0.01 to 5.0 mg/1 IAA. At 3.0 mg/1 IAA, in addition to leaf formation, 2% of the explants formed a rosette of leaves and 1% formed a shoot. With the highest level of IAA (5 mg/1), 2% of the explants formed a root. Expanding leaves 5 through 8 developed mostly into leaves without petioles on IAA and kinetin. Plant development occurred from 2% of the youngest primordial leaves on 0.03 mg/1 kinetin; otherwise, these primordia on 0.003 to 2 mg/1 kinetin developed into abnormal leaves. Primordia 3 and 4 developed into normal appearing leaves at levels of kinetin between 0.03 and 2 mg/1. At lower levels the leaves were abnormal.     

Immunohistochemical localization of IAA and ABP1 in strawberry shoot apexes during floral induction

By using an anti-indole-acetic acid (anti-IAA) monoclonal antibody and an anti-auxin-binding protein 1 (anti-ABP1) polyclonal antibody, IAA and ABP1 were immunohistochemically localized in strawberry (Fragaria ananassa Duch.) shoot apexes during floral induction. The spatial distribution patterns of endogenous IAA and ABP1 and their dynamic changes during floral induction were investigated. In addition, the affect of 1-N-naphthylphtalamic acid (NPA) on IAA distribution during floral induction was also analyzed. The results showed that IAA was present in the shoot apexes throughout the floral induction process, gradually concentrating in the shoot apical meristem (SAM). The distribution of ABP1 and its dynamic changes were similar to those of IAA. In addition, the ABP1 immune signal in SAM gradually increased as floral induction developed. On a morphological level, these results indicate both the spatial distribution and dynamic changes in endogenous IAA and ABP1 during the floral induction process. The close correlation found between IAA and ABP1 indicates that a cooperation occurs during the regulation of floral induction. The results also suggest that IAA was the significant agent for floral induction, and that SAM might be the place of the main action. Treatment with NPA during floral induction prevented the accumulation of IAA in the SAM, delayed the process of floral differentiation and induced an abnormal flower development. It is likely that IAA in the shoot apex is produced in young leaves and transported through the vascular tissues to the SAM and other places of function. Finally, an appropriate amount of IAA in the SAM and normal polar auxin transport are essential for floral induction and differentiation in strawberries.     

Stem fasciated, a recessive mutation in sunflower (Helianthus annuus), alters plant morphology and auxin level

BACKGROUND AND AIMS: Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus). METHODS: F1, F2 and F3 generations were obtained through reciprocal crosses between stf and normal plants. For the genetic analysis, a chi2 test was used. Phenotypic observations were made in field-grown and potted plants. A histological analysis of SAM, hypocotyl, epicotyl, stem and root apical meristem was also conducted. To evaluate the level of endogenous indole-3-acetic acid (IAA), a capillary gas chromatography-mass spectrometry-selected ion monitoring analysis was performed. KEY RESULTS: stf is controlled by a single nuclear recessive gene. stf plants are characterized by a dramatically increased number of leaves and vascular bundles in the stem, as well as by a shortened plastochron and an altered phyllotaxis pattern. By histological analysis, it was demonstrated that the stf phenotype is related to an enlarged vegetative SAM. Microscopy analysis of the mutant's apex also revealed an abnormal enlargement of nuclei in both central and peripheral zones and a disorganized distribution of cells in the L2 layer of the central zone. The stf mutant showed a high endogenous free IAA level, whereas auxin perception appeared normal. CONCLUSIONS: The observed phenotype and the high level of auxin detected in stf plants suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both SAM arrangement and hormonal homeostasis.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Auxin regulates the initiation and radial position of plant lateral organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical-basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical-basal position or the identity of the induced structures.