Brassinolide, a growth-promoting steroidal lactone

Brassinolide (BR), a naturally-occurring-steroidal lactone from rape ( Brassica napus L.) pollen, was compared with auxin for activity in a number of bioassay systems. Responses similar to IAA were elicited by BR in bioassays based upon bean hypocotyl hook opening, elongation of maize mesocotyl, pea epicotyl and azuki bean epicotyl sections, and fresh weight increase in Jerusalem artichoke (2,4-D used) and pea epicotyl sections. The azuki bean and dwarf pea epicotyl bioassays were much more responsive to BR than IAA (at 10 μ M ). Responses approximately two-fold greater in magnitude were elicited by IAA in the maize mesocotyl, bean hypocotyl hook and Jerusalem artichoke bioassays. Little or no response was elicited by BR (0.01 to μ M ) in the cress root or decapitated pea-lateral bud bioassays. A powerful synergism between BR and IAA was observed in the azuki bean, pea epicotyl and bean hypocotyl hook bioassays. Although, as previously reported, other steroidal substances are active in some of the bioassay systems tested, none compared with BR in magnitude and diversity of elicited responses.     

Longitudinal Gradients of Indole-3-Acetic Acid and Abscisic Acid in the Hypocotyl of Etiolated Bean Seedlings

The longitudinal distribution of abscisic acid (ABA) and indole-3-aceticacid (IAA) along the hypocotyl of 5-d-old etiolated Phaseolusvulgaris L. cv. Limburg seedlings was measured. IAA was analysedby the L-methyl-indole--pryone assay (2-MIP) and ABA by electroncapture gas chromatography (ECD-GC). Length and width of theinner parenchyma cells, growth rate and protein content werealso measured. Cell expansion occurred predominantly in a region20 mm below the centre of the hook where elongation rate wasmaximal and where protein concentration decreased rapidly withdistance from the hook. The ratio between ABA and IAA was constant along the lengthof the hypocotyl. On a fresh weight basis the concentrationof both growth substances was maximal in the upper (youngest)part, decreased in slightly older sections where cell expansionwas proceeding and was smallest in the basal regions where cellexpansion was complete. However, when expressed on a proteinbasis the concentration gradient of the hormones was the reverseof that described on a fresh weight basis. Key words: IAA, ABA, hypocotyl, etiolated, bean     

Roles of Abscisic Acid and Indoleacetic Acid in the Stunted Growth of Water-Stressed, Etiolated Squash Hypocotyls

The endogenous contents of indoleacetic acid (IAA) and abscisicacid (ABA) were assayed simultaneously in etiolated squash hypocotylsthat had been subjected to water stress applied to squash rootsby immersing them in 60 mM polyethylene glycol solution. Thegrowth rate and suction force of the hypocotyl under this water-stresscondition were determined for comparison with the two hormonecontents. Water stress retarded hypocotyl growth and increased the suctionforce of hypocotyl cells, as the ABA content increased, by 5-foldof the initial content one day after treatment with polyethyleneglycol. The ABA contents thereafter remained high up to day4. The increase in ABA content was detected in stressed hypocotylas early as 6 h after treatment, when water stress had not affectedhypocotyl growth. When the water stress was terminated one dayafter treatment began, hypocotyl growth immediately recovered,and the ABA content decreased to the value for unstressed hypocotylsin one day. The IAA content gradually decreased in unstressedhypocotyls, but was maintained at a high value in stressed hypocotyls. Logarithmic concentrations of endogenous ABA (ng/g fr wt) werecorrelated significantly with the suction force of the hypocotyls(r=0.92) and with the growth rates (r=–0.78). IAA contentwas correlated neither with the growth rate nor the suctionforce. These results suggest that the endogenous ABA contentwas associated with the stunted growth of the etiolated squashhypocotyls produced by water stress but that the endogenousIAA content did not have an important role in growth regulationunder water stress conditions. (Received April 11, 1984; Accepted October 2, 1984)     

Comparative Study on the Bioassay between N6, O2'-Dibutyryl Cyclic Adenosine 3,5-Monophosphate and Cytokinin

The physiological activity of diBcAMP by various classical bioassay methods of cytokinin was studied. It exhibits the cytokinin-like activity in many ways of biological response, such as: the unfolding of etiolated barley leaf, the expansion of radish cotyledon, the expansion and greening of cucumber cotyledon and the inhibition of the elongation of mung-bean hypocotyl, etc. The activity of 1 mM diBcAMP was approximately the same as the activity of 0.044 mM benzyladenine in the most of the tests. However, the activity of 1 mM diBcAMP was found to be higher than the optimal concentration (0.044 mM) of benzyledenine in the radish cotyledon under the expansion test. There was no toxicity of diBcAMP in all of the bioassay methods used, even with higher concentration.     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.     

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.     

Boron and blue light reduce responsiveness of <Emphasis Type="Italic">Arabidopsis</Emphasis> hypocotyls to exogenous auxins

We reported earlier that boron stimulates hypocotyl growth in several Arabidopsis ecotypes but not in the boron-deficient mutant bor1-1. Others have shown that boron influences the metabolism and transport of the plant hormone auxin. We investigated how boron, in interaction with light, influences Arabidopsis hypocotyl growth responses to the exogenous auxins 1-NAA, 2,4-D and IAA. In either light condition, 1-NAA similarly inhibited hypocotyl growth in bor1-1 and the corresponding WT (Col-0), while in both genotypes, boron did not essentially affect the extent of the inhibition. Whatever the light conditions and in the absence of boron, 2,4-D inhibited hypocotyl elongation in WT, while in BL seedlings, high responsiveness to 2,4-D vanished when boron was added to the culture medium. Hypocotyl of bor1-1 seedlings in all boron concentrations tested and grown in the dark or RL responded to the auxin similar to WT plants. In BL, the mutant hypocotyls retained full sensitivity to 2,4-D at 0.1 mM H₃BO₃ but lost that sensitivity by 2 mM. In both genotypes tested, in the dark or RL, IAA inhibited hypocotyl growth. Conversely, IAA stimulated hypocotyl elongation in both genotypes developed in BL at 0.1 mM H₃BO₃. That stimulation disappeared when the boron supply increased to 2 mM. Our results suggest that specifically in BL, boron reduces hypocotyl responsiveness to auxins 2,4-D or IAA via the functional transporter BOR1. Our results lead to a discussion of how BL and BOR1 influence the mechanisms of auxin transport into and out of the cell.     

Relationships between xanthoxin,phototropism, and elongation growth in the sunflower seedling Helianthus annuus L.

For phototropic curvature of a green sunflower seedling, only the hypocotyl has to be illuminated; the tip and cotyledons are not involved in stimulus perception. The etiolated seedling is phototropically insensitive, illumination of only the hypocotyl renders it sensitive. It is concluded that the photoreceptor is located within the responding organ. In curving seedlings, the endogenous indoleacetic acid (IAA) remains evenly distributed. However, the inhibitor, xanthoxin (Xa), accumulates on the illuminated side. The degree of phototropic response is generally related to the concentration of Xa. The amount of phototropic curvature is independent of the rate of elongation growth, the former can be changed without affecting the latter, and vice versa. The data conflict with the Cholodny-Went theory, whereas they support the hypothesis of Blaauw that the phototropic reaction is caused by the local accumulation of a growth-inhibiting substance on the irradiated side.Abbreviations CCC chlormequat, (2-chloroethyl)trimethylammonium chloride - GA₃ gibberellic acid - IAA indole-3-acetic acid - Xa xanthoxin     

Indoleacetic Acid and Molecular Differentiation Evidence for Interaction

Proteins of hypocotyls of bean were studied by electrophoresis. Proteins were extracted from hypocotyl segments of various stages of development starting with the relatively undifferentiated hook regions and proceeding by 2 cm segments down the hypocotyl. The proteins were the soluble (pH 7.4), the basic nuclear (histones), acidic ribonuclear and acidic chromosomal. Soluble proteins reflected differentiation of the hypocotyl in that lower hypocotyl segments had more different protein types than did the hook region. Indoleacetic acid (IAA) at 10^?6M when applied to the lower hypocotyl appeared to induce still more different proteins. However, at 10^?3M, IAA appeared to induce molecular dedifferentiation in that hypocotyl protein patterns began to resemble those of the hook. Histones also reflected differentiation, the hook having more histone types than the lower hypocotyl. IAA had no effect on histones. The hook region had two types of acidic chromosomal proteins, the lower hypocotyl one. When lower hypocotyl segments were incubated in 10^?3M IAA, the protein pattern resembled that of the hook in that the second protein normally present in the hook and not in the hypocotyl was in fact induced in the hypocotyl. The hook had two acidic ribonuclear proteins, the lower hypocotyl one. IAA did not affect this protein. These experiments suggest that IAA in some manner regulates molecular (protein) differentiation. It is further suggested that IAA accomplishes this control through the acidic nuclear proteins which are closely associated with genetic material and which reflect differentiation and are also affected by IAA.     

The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf.

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 microM were studied, with maximum IAA-induced curvature at 100 microM. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg 1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which it effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.     

Effect of isopropyl-N-(3-chlorophenyl) carbamate on the production of ethylene by mungbean hypocotyl sections

Isopropyl-N-(3-chlorophenyl) carbamate (CIPC) stimulates ethyleneproduction in hypocotyl sections of etiolated mungbean seedlings.The amount of ethylene produced is dependent on the concentrationsof CIPC applied. However, CIPC markedly inhibits IAA-inducedethylene production at relatively low concentration. It is possiblethat both CIPC and IAA are competing for the same cellular siteto induce ethylene production. However, the effectiveness ofCIPC in inducing ethylene production is much less compared toIAA treatment. The inhibition of ethylene production inducedby IAA at high concentration of CIPC may be solely due to thecomplete occupancy of the cellular site by CIPC and the effectof IAA is, therefore, eliminated. ¹ Present address: Mann Laboratory, University of California,Davis, California, U.S.A. (Received September 19, 1972; )     

Uptake and decarboxylation of indole-3-acetic acid during auxin-induced growth in lupin hypocotyl segments

The elongation growth of etiolated hypocotyl segments of lupin (Lupinus albus L.) was stimulated by acid pH (4.6 versus 6.5) and by IAA for periods of up to 4 h. After this time, the segments were unable to grow further. In the presence of an optimal IAA concentration (10 μM), acid pH increased the growth rate but had no effect on final growth. With suboptimal IAA (0.1 μM), however, acid pH increased growth in a more than additive way, suggesting a synergistic action between the two factors. This synergism may be explained by the increased IAA uptake and decarboxylation seen at an acid pH. These results reinforce the view that the effects of low pH and IAA on growth are not independent. Vanadate inhibited growth and also IAA uptake and decarboxylation. This inhibitor, therefore, probably inhibits growth not only by decreasing ATPase-mediated acidification but also by decreasing H⁺-dependent IAA uptake from the apoplasm. This dependence of IAA uptake on ATPase may be mediated by apoplasmic acidification. The amount of IAA decarboxylated increased when the assay conditions favored the growth of segments, indicating that IAA could be destroyed by decarboxylation during the auxin-induced growth.     

MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana

We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.     

Cell Wall Acidification and its Role in Auxin-Stimulated Growth

The role of cell wall acidification in auxin-stimulated growthwas examined in abraded hypocotyl segments of etiolated Cucumis.sativus seedlings. Acidification of the medium by these segmentswas strongly inhibited by a pretreatment and the continued presenceof 1?0 mol m^–3 vanadate, widely used as an inhibitor ofplasma membrane ATPase activity. Elongation of segments in pH6?5 buffer was almost completely inhibited by such a treatmentwith vanadate, and the promotion of growth by indole-3-aceticacid (IAA) seen in the absence of vanadate was completely abolished.However, both inhibited and uninhibited segments showed a pronouncedelongation in response to pH 4?0) buffer. In pH 4?0 buffer,in contrast to the results obtained at pH 6?5, IAA significantlypromoted growth in both the presence and absence of vanadate.The results indicate that IAA can promote growth in the absenceof endogenous acidification, but that an acid wall is necessaryfor wall loosening to occur. Key words: Acidification, auxin-stimulated growth, Cucumis sativus, vanadate     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

Variation in indole-3-acetic acid transport and its relationship with growth in etiolated lupin hypocotyls

The relationship between the variation in polar auxin transport (PAT) and elongating growth in etiolated Lupinus albus hypocotyls was investigated. Parameters of auxin transport, such as the amount transported, intensity of the transport and sensitivity to 1-N-naphthylphthalamic acid (NPA) inhibition were measured in isolated sections from different sites (apical, middle and basal) along the hypocotyls in seedlings of different ages. Auxin transport was studied by applying radioactive indole-3-acetic acid (IAA) to upright and inverted sections. Basipetal transport was much higher than acropetal and very sensitive to NPA inhibition, which indicates that transport is polarized. Polarity was expressed as the NPA-induced inhibition and the basipetal/acropetal ratio. As a rule, both the amount of IAA transported and the polarity varied with the age of the seedlings, with values increasing from 3 to 5d and then decreasing. Both parameters were higher in apical (where most growth is localized) than in middle and basal regions, although this longitudinal gradient tended to disappear with aging as hypocotyl growth slowed and finally ceased. The application of NPA did not modify hypocotyl elongation in 5-d-old intact seedlings. Derooting of the seedlings drastically reduced elongation in the control, while NPA partially restored the growth, which suggests that NPA induces an increase in auxin in the elongation region. These results suggest that a basipetally decreasing gradient in PAT along the hypocotyl, which changes with age, may be responsible for auxin distribution pattern controlling growth.     

In vivo metabolism of labelled indole-3-acetic acid during polar transport in etiolated hypocotyls of Lupinus albus: Relationship with growth

The in vivo metabolism of indole-3-acetic acid (IAA) in etiolated hypocotyls of lupin (Lupinus albus L., from Bari, Italy) was investigated by appliying IAA labelled with two radioisotopes ([1-¹⁴C]-IAA+[5-³H]-IAA) to the apical end of decapitated seedlings, followed by extraction of the radioactivity in the different regions along the hypocotyl. This method allowed detection of IAA decarboxylation in zones distant from the cut surface and, therefore, containing intact cells. When IAA was added directly in solution to the cut surface, decarboxylation was high especially in those hypocotyl regions where transient accumulations characteristic of the polar transport of IAA occurred. In 10-day-old seedlings such accumulations were observed both in the elongation zone (2^nd, 3^rd, and 4^th cm) and in the non elongating basal zone (8^th, 9^th and 10^th cm). When the IAA, instead, was applied with an agar block deposited on the cut surface, IAA metabolism (decarboxylation as well as conjugation) was increased but almost exclusively in tissues within 10 mm of the cut surface. In both kinds of experiment, the increase in IAA decarboxylation seemed to coincide with a decrease in the transport of IAA, since in the assay without agar the transient accumulations of radioactivity were probably due to a decrease in the transport velocity, while in the assay with agar the transport intensity was much lower than in the assay without agar. These results point to a competitive relationship between IAA metabolism and transport. Consequently, it is suggested that hypocotyl regions that probably use auxin for development processes (e.g., cell elongation and differentiation) may have a more intense IAA metabolism in parallel with their higher IAA concentrations.     

Isolation and expression of an elongation-dependent gene of mung bean (Vigna radiata) hypocotyl

A cDNA clone of an auxin up-regulated gene, ARG8 , was isolated from hypocotyl sections of etiolated mung bean [ Vigna radiata (L.) Wilczek] seedlings by differential screening. The deduced amino acid sequence suggested that ARG8 may encode a cell wall protein. The steady state mRNA level of ARG8 increased by treatment of hypocotyl sections not only with indole-3-acetic acid (IAA) but also with fusicoccin, and the auxin inducibility was inhibited by the addition of 0.3 M mannitol in the incubation medium. This indicated that it was not auxin but elongation that regulated the expression of ARG8 . The promoter activity of the 5'-flanking region of ARG8 was determined by assaying the transient expression of a luciferase fusion gene that was introduced into mung bean hypocotyl sections by the particle bombardment technique. The basal activity of the ARG8 upstream region was about a few tenths of that of a modified cauliflower mosaic virus 35S promoter, and it was increased a few fold by treatment with IAA. The auxin inducibility was completely suppressed by the addition of mannitol. A 5'-deletion analysis showed that a 53-bp region in the ARG8 promoter was important for the basal and elongation-dependent promoter activities.