Auxin Flow in Anther Filaments is Critical for Pollen Grain Development through Regulating Pollen Mitosis

It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10–12. Further studies employing the indoleacetic acid–lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [¹⁴C]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains.     

Reduction of indole‐3‐acetic acid methyltransferase activity compensates for high‐temperature male sterility in Arabidopsis

High temperature is a general stress factor that causes a decrease in crop yield. It has been shown that auxin application reduces the male sterility caused by exposure to higher temperatures. However, widespread application of a hormone with vast effects on plant physiology may be discouraged in many cases. Therefore, the generation of new plant varieties that locally enhance auxin in reproductive organs may represent an alternative strategy. We have explored the possibility of increasing indole‐3‐acetic acid (IAA) in ovaries by reducing IAA methyltransferase1 (IAMT1) activity in Arabidopsis thaliana. The iamt1 mutant showed increased auxin signalling in funiculi, which correlated with a higher growth rate of wild‐type pollen in contact with mutant ovaries and premature ovule fertilization. While the production of seeds per fruit was similar in the wild type and the mutant at 20 °C, exposure to 29 °C caused a more severe decrease in fertility in the wild type than in the mutant. Loss of IAMT1 activity was also associated with the production of more nodes after flowering and higher tolerance of the shoot apical meristem to higher temperatures. As a consequence, the productivity of the iamt1 mutant under higher temperatures was more than double of that of the wild type, with almost no apparent trade‐off.     

Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in <Emphasis Type="Italic">Torenia fournieri</Emphasis> L.

In the present report, we described the effects of indole-3-acetic acid (IAA), zeatin (ZT), gibberellin (GA₃), and abscisic acid (ABA) on in vitro pollen germination and pollen tube growth in Torenia fournieri L. The results showed that IAA and GA₃ stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and ZT had no significant effect on the process. The stimulating effect of exogenous IAA was particularly distinct, and led to synchronous growth of straighter and more slender pollen tubes compared with the controls. However, no significant changes were found in the germination of the treated pollen. The auxin efflux inhibitor, 10 μM 1-N-naphthylphthalamic acid (NPA), was also found to stimulate pollen tube growth. We measured the content of hormones (free IAA, ZT, GA₃, and ABA) in the stigmas and styles before and after pollination. The hormone contents of stigmas measured 0.5 h after pollination (0.5 HAP) showed that ABA content decreased, whereas the content of IAA, ZT, or GA₃ did not change significantly. The hormone level in pollinated styles (4 HAP) when pollen tubes had grown into the middle part of style was characterized by an increase in free IAA and GA₃ and a decrease in ABA, which was in agreement with the results that IAA and GA₃ promoted but ABA inhibited pollen tube growth in vitro. Furthermore, the change of IAA level in styles was most notable, which was accordant to the fact that auxin stimulated significantly pollen tube growth in vitro. Using immunoenzyme and immunogold labeling techniques and an anti-IAA monoclonal antibody, we confirmed that free IAA was present throughout style tissues, and distributed in the nucleus and cytoplasm of style cells. All these results suggested that hormones, especially IAA, play important roles in pollen tube growth of T. fournieri. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ORIGIN AND DEVELOPMENT OF POLLEN EMBRYOIDS AND POLLEN CALLUSES IN CULTURED ANTHER SEGMENTS OF HYOSCYAMUS NIGER (HENBANE)

The dedifferentiation of pollen grains of Hyoscyamus niger (henbane) into embryoids and calluses was examined by culturing identical segments of the same anther in a mineral salt-sucrose basal medium and in the basal medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid, respectively. Addition of auxin enhanced anther efficiency but did not affect the number of embryogenic pollen grains of an anther segment transformed into calluses. In anther segments cultured in the basal medium, the organogenetic part of the pollen embryoid was formed by the division of the generative cell alone, or by the division of both generative and vegetative cells. More or less similar pathways were followed by pollen grains of anther segments cultured in a medium containing auxin to form calluses. Culture of anther segments in a medium containing a high concentration of auxin (50.0 mg/l) led to a significant reduction in the yield of calluses which were formed almost entirely by the division of both generative and vegetative cells. The bearing of these observations on the role of auxin in determining the pathway of differentiation of embryogenic pollen grains in cultured anther segments is considered. The appearance of embryogenic pollen grains in close proximity to the tapetum as seen in longitudinal sections of cultured anther segments has suggested a role for a gradient of tapetal factors in embryogenic induction.     

Auxin levels,antiauxin(s) and androgenic plantlet formation in isolated pollen cultures ofNicotiana tabacum

Summary Anthers ofNicotiana tabacum var. Badischer Burley contain endogenous auxins, one of these was identified as indoleacetic acid. At the developmental stage shortly after the first pollen mitosis the anthers contain equivalents of 0.1 mg IAA per kg fresh weight. This endogenous auxin level is maintained during the eight-day preculture of the anthers prior to isolated pollen culture. However, in anthers of short-day plants, which are characterized by a high proportion of embryogenic pollen at the end of preculture (Heberle-Bors andReinert 1979), an increase of the auxin level till the fourth day of culture is detectable.Preculture of anthers in the presence of an inhibitor of auxin synthesis (7-azaindole) and an antiauxin (-(o-chlorophenoxy)-isobutyric acid) results in enhanced plantlet yield by pollen cultures. The significance of these observations for androgenesis is discussed.     

The short-rooted vitamin B<Subscript>6</Subscript>-deficient mutant <Emphasis Type="Italic">pdx1</Emphasis> has impaired local auxin biosynthesis

The phytohormone auxin regulates many aspects of plant growth and development. Auxin often acts distantly from the site of its biosynthesis and this long-distance-transported auxin is well known to play a critical role in eliciting physiological responses including regulating root development. Auxin can be produced in roots, yet the function of locally synthesized auxin in root growth is unclear. The major auxin in plants, indole 3-acetic acid (IAA), is mainly synthesized through tryptophan (Trp)-dependent pathways that require pyridoxal phosphate (an active form of vitamin B₆)-dependent enzymes. We previously reported that the Arabidopsis vitamin B₆ biosynthesis mutant pdx1 has stunted root growth although the underlying cause is unknown. Here we showed that the pdx1 root is deficient in auxin biosynthesis. By reciprocal grafting of pdx1 and the wild type, we demonstrated that the stunted root growth in pdx1 is caused by a locally generated signal(s) in roots. To test whether auxin might be one such signal, the auxin responsive DR5::GUS reporter was introduced into the mutant. The DR5::GUS activity in pdx1 root tips was greatly reduced compared with that in the wild type although the auxin response was unaltered. pdx1 also suppresses the root hair growth defects in the auxin overproduction mutant yucca. These data indicate that pdx1 is impaired in Trp-dependent auxin biosynthesis, which may contribute to the short-root phenotype of pdx1. We suggest that locally synthesized auxin may play a critical role in postembryonic root growth.     

Isoenzyme Polymorphism in Flowering Plants I. Diffusion of Enzymes Out of Intact Pollen Grains

Esterases, leucine aminopeptidases, catalases, amylases and acid phosphatases diffuse out of intact and ungerminated pollen grains of Oenothera organensis, whether suspended in 1 % sodium chloride or in pollen medium. A total of 15 esterase isozymes are recorded; 5 of them appear within 5 minutes, 8 within 30 minutes, 9 within 2 hours, and 13 within 19 hours. Pollen grains suspended for 19 hours gave much stronger isozyme bands than macerated pollen grains. However, one esterase hand was consistently missing from the 19 hour suspensions, although present in all others. It is suggested as a working hypothesis that the early growth of pollen tubes and possibly even germination of pollen grains may be influenced by the metabolic products of pistillate tissues caused by the diffused pollen enzymes, and that inactivation of these enzymes by stigmatic or stylar components could lead to incompatibility reactions.     

Role of auxin in regulating Arabidopsis flower development

To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip. Electronic Supplementary Material Supplementary material is available for this article at This paper is dedicated to Orna Aloni for continuous support and management over many years.     

On the Relation Between the Effects of Auxin on Growth, pH and Potassium Transport

The relation between the effects of auxin on growth, pH and potassium transport in hypocotyl segments of Helianthus annuus was studied. In a solution containing 20 mM Na₂SO₄ auxin-induced growth was accompanied by an auxin-induced pH drop in the medium. (NH₄)₂SO₄, at the same concentration, brought about an almost complete abolishment of the effect of auxin on the pH. The magnitude of auxin-induced growth was, however, only slightly reduced. This result does not confirm the hypothesis according to which the action of auxin on growth is a result of its effect on the pH. In a solution containing 2 mM sodium phosphate buffer an inhibitory action of IAA on the release of potassium from the tissue was observed. Addition of 20 mM Na₂SO₄ to the medium brought about a complete abolishment of this effect. The magnitude of auxin induced pH drop was, however, similar in the two treatments. It was concluded that, although under suitable experimental conditions, a close relationship may exist between the effects of auxin on pH on K⁺ transport, the coupling between the two phenomena is not obligatory.     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pollen embryogenesis in anther cultures of Solanum carolinense L.

The direct differentiation of bicellular pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and plantlets was induced by culturing whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency of embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was similar to that of zygotic embryos. In a previous study, it was show that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. It appears then that there are two distinct pathways of androgenesis in this species that are determined by the type of auxin present in the medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BA benzyladenine - KIN kinetin - MS Murashige and Skoog     

The auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway

 Hypocotyls of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) do not elongate in response to exogenous auxin, but can respond to gravity. This appears paradoxical in light of the Cholodny-Went hypothesis, which states that shoot gravicurvature results from asymmetric stimulation of elongation by auxin. While light-grown dgt seedlings can achieve correct gravitropic reorientation, the response is slow compared to wild-type seedlings. The sensitivity of dgt seedlings to inhibition of gravicurvature by immersion in auxin or auxin-transport inhibitors is similar to that of wild-type plants, indicating that both an auxin gradient and auxin transport are required for the gravitropic response and that auxin uptake, efflux, and at least one auxin receptor are functional in dgt. Furthermore, dgt gravicurvature is the result of asymmetrically increased elongation as would be expected for an auxin-mediated response. Our results suggest differences between elongation in response to exogenous auxin (absent in dgt) and elongation in response to gravistimulation (present but attenuated in dgt) and confirm the presence of two phases during the gravitropic response, both of which are dependent on functional auxin transport. Received: 16 July 1999 / Accepted: 24 September 1999     

The auxin transporter,OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.)

Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root (PR) and lateral root (LR) development, and almost nothing is known about how auxin and Cd interfere with root hair (RH) development. Here, we characterize rice osaux1 mutants that have a longer PR and shorter RHs in hydroponic culture, and that are more sensitive to Cd stress compared to wild‐type (Dongjin). OsAUX1 expression in root hair cells is different from that of its paralogous gene, AtAUX1, which is expressed in non‐hair cells. However, OsAUX1, like AtAUX1, localizes at the plasma membrane and appears to function as an auxin tranporter. Decreased auxin distribution and contents in the osaux1 mutant result in reduction of OsCyCB1;1 expression and shortened PRs, LRs and RHs under Cd stress, but may be rescued by treatment with the membrane‐permeable auxin 1‐naphthalene acetic acid. Treatment with the auxin transport inhibitors 1‐naphthoxyacetic acid and N‐1‐naphthylphthalamic acid increased the Cd sensitivity of WT rice. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species‐mediated damage was enhanced, further increasing the sensitivity of the osaux1 mutant to Cd stress. Taken together, our results indicate that OsAUX1 plays an important role in root development and in responses to Cd stress.     

THE EFFECT OF DELPHINIUM NELSONII POLLEN ON SEED SET IN IPOMOPSIS AGGREGATA,A COMPETITOR FOR HUMMINGBIRD POLLINATION

Sympatric plant species can compete for pollination services in several ways. For example, pollinators may move between species and deposit heterospecific pollen on stigmas, which in turn may reduce the efficacy of conspecific pollen. We explored this possibility by determining the effect of Delphinium nelsonii pollen on seed set in Ipomopsis aggregata. These montane herbs are pollinated by hummingbirds, experience heterospecific pollen deposition in nature, and suffer reduced seed set in each other's presence. We hand-pollinated flowers of I. aggregata with either pure conspecific pollen or a mixture of pollen of the two species. Resulting pollen loads on stigmas ranged from 0–865 D. nelsonii grains and from 10–336 I. aggregata grains; mean seed set per flower was 11.3. There was no detectable effect of D. nelsonii pollen load on I. aggregata seed set. It is possible that seed set reductions seen in previous studies of competition for pollination between these species were caused by pollen wastage, pollen layering on the pollinator, or the temporal sequence of pollen arrival at the stigma.     

Root Nodule Symbiosis II

Nodule-roots of Myrica cerifera (Southern Wax Myrtle) and Casuarina cunning hamiana (Australian Pine) have a negative geotropic curvature. Studies of their endogenotts auxin content revealed a pattern of correlation: the absence of detectable auxin when the geotropisni was negative. Non-nodulated roots of Myrica exhibited a normal positive geotropic curvature and possessed an auxin content within an anticipated range (10 mg/kg). Root nodules of Alnus species, whose roots exhibit a positive geotropic curvature, contained measurable endogenous auxin (20 mg IAA/kg). The presence of an indoleaectic acid oxidase system in Myrica and Casuarina root nodules has heen described and correlations are drawn between non-detectable endogenous auxin concentrations and high enzymatic activities. It is suggested that the negative geotropic curvature of the nodule-roots of Myrica and Casuarina is due to the presence of a sub-optimal concentration of auxin which in turn results from the activity of an indoleacetic aeid destroying system.     

Relazioni ormonali nello sviluppo dell'ovario delle orchidee i. effetto in vivo del trattamento con auxine

Abstract

HORMONAL RELATIONS IN THE DEVELOPMENT OF ORCHID OVARY. I. - THE EFFECTS OF TREATMENTS WITH AUXINS ON THE INTACT PLANT. — The flowers of different species of Orchids have been treated with auxins, pure or in solutions, and with pollen from the same or a different species. The auxin treatment consistently induces the elongation of the column and the growth of the ovary. The treatment with the most active of the auxins used (pure NAA) induces necrosis of the column; in this case, growth of the ovary can last for more than one year, till ripening of the fruit. Morever, the interruption of development at any stage, is more frequent.

The stimulation of the ovary can be obtained also with the introduction of auxin into wounds at the basis of the column. In Zygopetalum NAA induces parthenocarpy but not adventive embriony which is induced with exogenous pollen.     

Is there a role for auxin in early embryogenesis?

The very young embryo of a flowering plant is not an idealsystem in which to study the effects of auxin. Conversely, auxin isusually not considered as a major component of developmental processesin early embryogenesis. However, recent findings from both experimentalstudies in Brassica and analyses of developmental mutants inArabidopsis make it worthwhile to examine critically thepossibility that auxin may have a role in early embryogenesis. In thisreview, we will focus on specific processes, such as formation of anapical-basal axis of polarity and the initiation of the primary rootmeristem. To provide a conceptual framework in which to discuss possibleeffects of auxin, we will first briefly summarise essential features ofearly embryogenesis in Arabidopsis. This will be followed by anevaluation of relevant data suggesting a role for auxin in axisformation and root meristem initiation. Finally, we will discuss a fewexperimental approaches that we believe are necessary to examine whetheror not auxin plays a role in fundamental processes of earlyembryogenesis.