EMASCULATION EFFECTS ON FILAMENT GROWTH IN IPOMOEA NIL (CONVOLVULACEAE)

Filament growth in the morning glory Ipomoea nil is promoted by gibberellic acid (GA₃) and inhibited by ethylene production during the earlier stages in development (Koning and Raab, 1987). The effects of anther removal on filament growth were examined in I. nil. Three-quarters of the calyx and corolla tissues were removed in order to emasculate the stamens. This removal caused filament growth to be inhibited in the intact stamens (69 hr before anthesis) as well as the emasculated stamens (69 to 21 hr before anthesis). When the ethylene biosynthesis inhibitors CoCl₂ and aminoethoxyvinylglycine (AVG) were applied in separate experiments (to eliminate wound ethylene generated by the damaged tissues), filament growth was promoted to control levels observed in intact flower buds (69 hr before anthesis). Our data suggest that the wounding effect from the calyx and corolla tissues and subsequent reduction in filament growth override any observable effects with anther removal on filament growth. Apparently, the removal of the calyx and corolla tissues severs a normal hormonal relationship between these floral organs and the developing filament.     

Role of plant growth substances in MS33-controlled stamen filament growth in Arabidopsis

The rapid growth of stamen filaments just before flower anthesis in Arabidopsis thaliana does not occur in the male sterile33 ( ms33 , formerly known as msZ ) mutant. ms33 filaments were approximately 40% shorter than the wild type (WT), and there was corresponding reduction in the epidermal cell length of filaments. This suggests that MS33 controls the final cell-elongation phase of filament growth. Both low temperatures and gibberellic acid (GA₃) restored filament and cell growth in intact ms33 flowers, but these treatments only had a small promotive effect on WT filaments. Decapitation experiments involving the removal of the anther had the opposite effect on WT and ms33 filaments; growth was inhibited in WT, but was increased in ms33 filaments. In young stamen primordia cultured in vitro, filament growth was less in WT, but more in ms33 , than in respective in vivo produced filaments. Plant growth substances (PGSs), GA₃ and indole-3-acetic acid (IAA) were promotive, zeatin had no effect, and abscisic acid (ABA) and ethrel inhibited filament growth in both intact and decapitated WT and ms33 filaments. Together these observations suggest that MS33 is activated immediately before anthesis and that the MS33 product either regulates temporal biosynthesis of gibberellins (GAs) and/or IAA or makes the filament tissue sensitive to these PGSs, which in turn trigger cell elongation and filament growth. The data also suggest that ms33 mutant anthers contain a relatively high ratio of growth inhibitors to promoters, which inhibits epidermal cell elongation and filament growth.     

The mechanism of stamen movement in <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (Calycanthaceae): differential cell growth rates on the adaxial and abaxial surfaces of filaments after flower opening

In flowers of Chimonanthus (Calycanthaceae), stamen movements have been known for 150 years. Stamens are initially recurved when flowers open. Subsequently, within approximately 48 h, the stamens gradually turn upright and eventually enclose the gynoecium. However, the exact mechanism involved in this process is still unresolved. In this study, we found that in recurved stamens at the female stage the cell lengths in the epidermal and hypodermal tissue of the abaxial surfaces were significantly smaller than those of the adaxial surfaces. In erect stamens, however, no significant difference in cell length between the hypodermal tissues of both surfaces was found. Although the difference in cell length between the epidermal tissues for erect stamens was significant, it was smaller than that in recurved filaments. We found no variations in cell number between the epidermal tissues or between the hypodermal tissues among recurved and erect stamens. Thus, we conclude that in Chimonanthus the differential cell growth rates between the adaxial and abaxial surfaces of filaments could account for the gradual inward stamen movement following flower opening rather than cell division. Furthermore, application of indole-3-acetic acid (IAA) and gibberellic acid (GA₃) to intact flowers in vitro or the wounding of stigmas significantly promoted the stamen erection rate. Surprisingly, we did not observe any effect on this movement following hand-pollination. Different patterns of stamen movement in Calycanthaceae and their implications for reproductive biology are also discussed.     

Endogenous Levels and Transport of 1-Aminocyclopropane-1-Carboxylic Acid in Stamens of Ipomoea nil (Convolvulaceae)

Filament and corolla growth in flowers of Ipomoea nil are inhibited by ethylene production. Anthers inhibited filament growth in vitro during younger stages of development even in the presence of the growth promoter gibberellic acid (GA₃). To test whether the anthers could be sources of 1-aminocyclopropane-1-carboxylic acid (ACC) endogenous levels of ACC and ethylene production were monitored using gas chromatography. To also test whether the filaments could be transport vectors for ACC the movement of [¹⁴C]ACC was assessed by scintillation counting from donor agarose blocks, through filament sections, and into receiver agarose blocks. While ACC levels fluctuated in anthers 87 to 21 h before anthesis, anthers contained increased levels of ACC from 15 to 6 hours before anthesis. Ethylene production also fluctuated but peak levels were shifted about 6 hours closer to anthesis than ACC levels within the anthers. Both ACC and ethylene levels in filaments showed fluctuations similar to those in the anthers. [¹⁴C]ACC movement became increasingly basipetal during development. Older stages showed greater polar [¹⁴C]ACC efflux rates, while all stages showed constant polar influx rates. Low levels of endogenous ACC were transported basipetally from the anther through the filament into agarose blocks at all stages of development. Corresponding levels of endogenous ethylene production remained constant between the various stages during ACC transport. We have evidence that stamens of I. nil have a role as source tissues and transport vectors for ACC, to stimulate corolla growth, such as corolla unfolding and senescence.     

The role of gibberellins in the growth of floral organs of Mirabilis jalapa

Evidence that the anther is a source of GAs which may be a mainfactor limiting the growth of floral organs of Mirabilis jalapais presented. GAs in floral tubes at different developmentalstages were surveyed using TLC followed by bioassay with ‘Tan-ginbozu’dwarf rice seedlings. The amount of GA_s in the floral tube increasedrapidly after the calyx became visible above the bract 8 daysbefore anthesis, reached a maximum 5 days before anthesis, thendeclined markedly thereafter. The stamen had relatively largeamounts of GAs and mainly accounted for the change of GA_s duringthe flower development. The GA content in the anther was 3 timesas much as that in the filament. Exogenously supplied GA3 promotedelongation of sections in excised flower organs. Extracts ofanthers also showed definite growth promotion of style sections.Removal of the anthers greatly reduced the final length of thefloral tube. (Received December 7, 1974; )     

ROLE OF GROWTH SUBSTANCES IN THE FILAMENT GROWTH OF FUCHSIA HYBRIDA CV “BRILLIANT”

Filaments of Fuchsia hybrida cv “Brilliant” double in length within 24 hr after bud opening. Filament growth characterized by fresh wt increase and cell elongation was significantly inhibited in vitro by l-aminocyclopropane-l-carboxylic acid (ACC) but was not promoted by any growth regulator tested. Ions of Co²⁺ blocked the inhibitive effects of ACC in vitro suggesting that ethylene produced from ACC is the growth inhibiting substance. Ethylene levels surrounding the filaments within the closed bud decreased during development, and premature opening of the sepals which released the ethylene into the atmosphere resulted in rapid filament growth. The ACC levels were found to be much higher in the anthers than the filaments. This suggests that ethylene produced from floral organs other than filaments regulates filament elongation in Fuchsia. This is the first report of filament growth which cannot be promoted by application of growth regulators but which is inhibited by ethylene.     

Gibberellin response in lettuce hypocotyl sections

Excised lettuce (Lactuca sativa L.) hypocotyl sections retain the ability to elongate in response to gibberellic acid (GA₃) addition. In 48 hr at 30 C a GA₃-treated segment more than doubles while a control segment elongates less than 50%. Auxin has no detectable effect on this system. Sensitivity to GA₃ is not decreased by apex or root removal. Of the experimental variables tested, temperature, sucrose, and preincubation in water affect growth both with and without GA₃. Blue and far red light inhibit growth without GA₃; this inhibition is reversed by GA₃. Potassium chloride stimulates growth of illuminated sections treated with GA₃ but has no effect on control growth. When sections are incubated in the dark, KCl has a promotive effect on elongation.     

AN ANALYSIS OF STAMEN FILAMENT GROWTH OF NIGELLA HISPANICA

The post-meiotic stamen filament of Nigella hispanica L. under greenhouse conditions grows in length from 1 mm to approximately 10 mm at maturity in 16 days. Analysis of the filament epidermis suggests that the intercalary meristem is diffuse along the filament with a mid-point of activity near the center of the filament. The point of maximal activity, while initially central, is variable as cell division nears completion. Measurement of cell lengths along filaments suggests that an elongation gradient from base to tip is operative in filaments 1 mm and longer. Average cell lengths of epidermal cells increase faster than do those of terminal cells. Once average cell length begins to increase in any region of the epidermis it continues to do so until flower maturity. At maturity the longest epidermal cells are near the filament base and the shortest cells are at the tip. The differences between cell division and cell elongation patterns suggest that these two processes are controlled by different sites or substances. A comparison is made between the development of the Nigella filament and other determinate organs having intercalary meristems.     

Regulation of flower development in cultured ears of maize (Zea mays L.)

Summary Young ears of maize were cultured in two different liquid media containing either kinetin (KN) or kinetin + gibberellic acid (KN + GA₃) in order to manipulate stamen and gynoecium development. In KN medium, stamens developed and gynoecia aborted in the flowers of the cultured immature ears. In the KN + GA₃ medium, however, ovaries with silks developed and stamens aborted. These differential morphological events were recorded with SEM photomicrographs at regular intervals after excision of ear inflorescences. In addition, the mitotic activity in the developing or aborting organs was determined over a 75-h period. It increased from 6% to 14% in developing organs (i.e. stamens in KN medium, and gynoecia in KN + GA₃ medium) and gradually decreased to 1% in the degenerating organs (i.e. gynoecia in KN medium, and stamens in KN + GA₃ medium) by 45 h of culture. The mitotic activity reached zero in degenerating flower organs by 75 h of culture. Whether these differential sensitivities to the exogenously applied members of these two plant growth regulator classes are unique to our in vitro system or reflect a more general control feature of in vivo inflorescences must await further clarification.     

Stamens and Gibberellic Acid in the Regulation of Flavonoid Gene Expression in the Corolla of Petunia hybrida

Stamen removal at an early stage of flower development inhibits anthocyanin synthesis and chalcone flavanon isomerase (CHI) enzyme activity in corollas of Petunia hybrida. The inhibition can be overcome by gibberellic acid (GA₃) application. Gibberellin also induces anthocyanin synthesis in detached, young green corollas, grown in vitro in a sucrose medium and promotes CHI enzyme activity. Western blot analysis indicates an increase in chalcone synthase (CHS) and CHI protein levels following GA₃ treatment in both the in vivo and the in vitro systems. Northern blot analysis shows a higher level of steady-state mRNAs for CHS and CHI 24 hours after GA₃ application. In corollas from a transgenic plant containing a β-glucuronidase gene driven by a CHI promoter, a sixfold increase of β-glucuronidase activity was measured following GA₃ application. The mode of action of stamens and GA₃ control over flavonoid gene expression is discussed.     

FURTHER OBSERVATIONS ON THE DIFFERENTIAL SENSITIVITIES TO PLANT GROWTH REGULATORS BY CULTURED “SINGLE” AND “DOUBLE” FLOWER BUDS OF NIGELLA DAMASCENA L. (RANUNCULACEAE)

In Nigella damascena, where “doubleness” behaves as a single gene character, recessive to the normal wild “single” form, we detected a number of differential responses of cultured floral apices of the two forms to plant growth regulators. “Single” buds initiated all organ types on a defined medium lacking any plant growth regulators. “Double” buds, however, required either kinetin or GA₃ for the development of a normal organ complement. Kinetin enhanced the growth and development of buds of both genotypes, whereas GA₃, at all concentrations, specifically inhibited the initiation and growth of stamens and nectaries in “single” buds. No combination of IAA or kinetin overcame this inhibition of “single” buds by GA₃. In the presence of both IAA and GA₃, “single” buds, following the initiation of normal carpels, initiated a few abnormal carpelloid structures from regions which had failed to produce stamens and nectaries. These observations, coupled with other studies including those on the endogenous levels of gibberellin in the two genotypes, suggest that “doubleness” correlates strongly with a modification of gibberellin metabolism. The causal connection between the two phenomena is as yet to be discovered.     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

HISTOGENESIS OF THE ANDROECIUM AND GYNOECIUM IN DOWNINGIA BACIGALUPII

A histogenetic investigation of the synandrous androecium and syncarpous gynoecium in the flower of Downingia bacigalupii Weiler (Campanulaceae; Lobelioideae) was undertaken for the purpose of comparing the modes of initiation, early growth and fusion in these floral whorls with that reported previously for the perianth in this species. Stamens are initiated as separate organs from the second tunica layer and underlying corpus regions of the concave floral meristem. Subsequent growth of stamens involves apical and intercalary growth in length and rudimentary marginal growth in breadth. Tissues of the four microsporangia originate from hypodermal sporangial initial cells and the filament is formed by intercalary growth at the base of the anther. Lateral fusion of stamens is ontogenetic and involves cuticular fusion of adjacent epidermal layers. The two emergent carpel primordia arise as crescentic organs by periclinal divisions in the second tunica layer and corpus zones. Carpel primordia also undergo apical and intercalary growth in length as well as extensive marginal growth in breadth. Radial growth in carpels is mediated by an adaxial meristem which shows its greatest concentration of activity at the carpel margins. Carpel fusion appears to be partially ontogenetic accompanied by zonal growth. Closure of the stylar canal is by the formation of a transmitting tissue derived from the protodermal layers of the adaxial carpel surfaces. A discoid nectary is initiated around the base of the style and formation of the inferior ovary is by intercalary growth of the base of the concave floral bud. The two parietal placentae originate as longitudinal outgrowths from the walls of the floral cup. Ovule initiation is simultaneous at first and then intercalary during subsequent elongation of the ovary. The ovules are anatropous, unitegmic and tenuinucellate. Stamen and carpel procambium shows a slight delay in differentiation when compared to that reported for the perianth and bract, but in all other respects carpels resemble other floral organs in their patterns of histogenesis and early growth. Stamens diverge from the other floral organs in their early pattern of growth, but a consideration of all features of their histogenesis suggests an appendicular rather than an axial interpretation of these organs.     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

Morphogenetic Effects on Vegetative Plants of Poa pratensis L. of 6-Azauracil, (2-Chloroethyl)- phosphonic acid, and (2-Chloroethyl) trimethyl-ammonium chloride and their Interaction with Gibberellic Acid

Fresh and dry weights and leaf size of Poa pratensis were reducedwhen treated with 6-azauracil (AzU), (2-chloroethyl)phosphonicacid (CEPA), or (2-chloroethyl)trimethylammonium chloride (CCC).AzU and CEPA inhibited epidermal cell division without inhibitingcell elongation, while CCC inhibited mainly cell elongationand cell division to a small extent. The ratio of blade lengthto sheath length and the blade length/width ratio were reduced,but leaf emergence and tillering were increased by AzU and CEPA.CCC affected only the latter three features. Like GA₃, CEPAinduced stem formation, but internodes were shorter. GA₃ was ineffective in preventing leaf-growth inhibition byAzU, which inhibited Ga₃-induced cell elongation. The inhibitoryeffect of CEPA on leaf growth was apparently reversed by GA₃,but this was due solely to increased cell elongation, the reductionin cell number being unaffected. Ga₃ reversed the effect ofCCC on leaf length, as well as on cell size and number. Simultaneousapplication of the inhibitors produced a complex interactionin reducing leaf length and number and size of epidermis cells.It is postulated that AzU, CEPA, and CCC have different modesof action because they have specific effects on plant growthand different effects on GA₃-induced cell elongation.     

Effect of gibberellins on growth of pea seedling internode

Elongation of internode segments of dwarf pea seedlings excised 4 mm below the plumular hook was stimulated by GA₃ but not by GA₁ or GA₅. However, all three gibberellins induced cell elongation in the region from which this segment was isolated on application to intact seedlings. It is concluded that GA₁ and GA₅ are converted to a GA₃-like hormone. Measurement of epidermal cell elongation in the epicotyl further indicates that GA₃ or a GA₃-like hormone may be the functional form of the hormone required for cell elongation.     

Comparison of endogenous gibberellins and of the fate of applied radioactive gibberellin a(1) in a normal and a dwarf strain of Japanese morning glory

The effect of application of GA₃ on hypocotyl growth, the endogenous GAs, and the metabolism of applied ³H-GA₁ were investigated in relation to dwarfism and light-mediated growth inhibition in the normal (tall) strain Violet and the dwarf strain Kidachi of Japanese morning glory (Pharbitis nil). GA₃ applied in a wide concentration range (10⁻⁹ to 10⁻³m) to 4-day-old seedlings caused great extension of the hypocotyls in light-grown plants of both the normal and the dwarf strain. However, the dwarf strain did not attain the same length as the normal one at any given GA₃ concentration, even when saturation was reached. Dark-grown plants of the dwarf strain responded to GA₃, although relatively much less than light-grown ones; dark-grown plants of the normal strain showed no GA₃ response at all.     

Genotypic variation in the responsiveness to GA3 within tall (rht1) and semi-dwarf (Rht1) spring wheat

The effect of GA₃ on coleoptile-and first leaf elongation of tall (rht1) and semi-dwarf (Rht1) nearly-isogenic genotypes, within each of 25 random F₉ wheat families, was determined on seedlings grown in a growth room at 18 °C. Conspicuous and very significant inter-family variation in the response of the first leaf to GA₃ application was found in both the rht1 and Rht1 genotypes. The magnitudes of the response of the different families within genotypes to GA₃ were not related to the leaf length of their untreated seedlings. It is suggested that, under given environmental conditions, background genotypic effects, inducing inter-family variation in responsiveness to GA₃, regulate the elongation growth up to the limits set by the Rht alleles.     

Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hybrida

Removal of stamens, or even of only the anthers, at an early stage of corolla development, before the start of main anthocyanin production, inhibited both growth and pigmentation of attached corollas of Petunia. When only one or two stamens were removed from one side, the inhibition was restricted to the corolla side adjacent to the detached stamens. Application of gibberellic acid (GA₃) substituted for the stamens in its effect on both growth and pigmentation. In detached corollas, isolated at the early-green stage and grown in vitro in sucrose medium, GA₃ promoted growth and was essential for anthocyanin synthesis. A marked enhancement of anthocyanin production was observed 48 h before the increase in corolla growth rate. Corollas detached at later stages were able to continue their growth and pigmentation in sucrose without GA₃. When Paclobutrazol (-[(4-chlorophenyl)-ethyl]-(1,1-dimethylethyl)-H-1,2,4-triazol-1-ethanol), an inhibitor of gibberellin biosynthesis, was added to the growth medium of in-vitro-grown corollas, pigmentation was inhibited but there was no effect on corolla growth. Low levels of GA₃ counteracted the Paclobutrazol effect on pigmentation but did not affect growth. The above results indicate that the effect of GA₃ (and probably that of the stamens) on corolla growth is independent of its effect on pigmentation. Gibberellic acid and paclobutrazol had no effect on [¹⁴C]sucrose uptake by in-vitro-grown corollas. The activity of phenylalanine ammonialyase was correlated with the effect of stamens and GA₃ on pigmentation in corollas grown in vivo and in vitro.Abbreviations GA gibberellin - GA₃ gibberellic acid - PAC Paclobutrazol - PAL phenylalanine ammonia-lyase     

Studies on Flowering Responses of Urena lobata

Urena lobata L. is a qualitative short-day plant with a critical dark period of about 12 hr. A minimum of 12 short days are required for floral initiation. Gibberellic acid (GA₃) sprayed onto the leaves promoted flowering. The growth retardant, (2-chloroethyl)-trimethylammonium chloride (CCC) markedly inhibited flowering and stem elongation. This inhibition was reversed by subsequent application of GA₃.