Free Indole-3-acetic Acid Research Articles

Influence of the Indoleacetic Acid‐Lysine Synthetase Gene (iaaL) of Pseudomonas syringae subsp. savastanoi on Yield Attributes of Potatoes

AbstractThe iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid‐lysine synthetase which conjugates free indoleacetic acid (IAA) with lysine. lAA‐lys is biologically less active than free IAA. The iaaL coding region was expressed under the control of the cauliflower mosaic virus 35S promoter and transgenic potato plants were produced (Spena et al. 1991). 35S iaaL potato plants are characterized by increased internodal length and epinastic bending of older leaves. In three greenhouse experiments with plants grown in pots of different size and in two growth chamber experiments tuber number increased in iaaL transgenic plants compared to untransformed and vector‐transformed controls of the same genotype. The increase in tuber numbers observed under controlled conditions was reflected in tuber yield which increased in the pot grown transgenics.

Compartmentation of indole-3-acetic acid metabolism in protoplasts isolated from leaves of wild-type and IAA-overproducing transgenic tobacco plants

Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [15N1]IAA synthesised from [15N1]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SRI plants, and in IAA-overproducing plants expressing the Agrobacterium tumefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was located exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conversion into IAA by action of the iaaH-encoded hydrolase. The free-IAA level, however, was raised only 8% in iaaM/iaaH compared to iaaM protoplasts, whereas the level of IAA-conjugates was increased more than fivefold. For both genotypes, the location of IAA conjugates was restricted to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion to the strong cauliflower mosaic virus (CaMV) 35S promoter. Compared with the wildtype, this led to an 18-fold higher conversion of [15N1]tryptophan to [15N1]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protoplasts. Also in these genotypes, IAA conjugates were exclusively cytosolic. There was no major difference between transgenic and wildtype protoplasts in the proportion of chloroplastic to total cellular IAA, although the chloroplastic IAA and [15N1IAA pools in the transformant were threefold and eightfold higher, respectively. Since the IAM pool in transgenic plants is exclusively cytosolic, these findings suggest that the increased chloroplastic [15N1IAA pool in 35S-iaaM/iaaH protoplasts is synthesised in the cytosol but rapidly transported into the chloroplast. Furthermore, the presence of IAA in the chloroplast together with the exclusively cytosolic location of IAA conjugates, suggests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxylative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 562–568), whereas the second is located in the chloroplast and is seemingly not directly regulated inside the organelle by either of these two processes. The cytosolic IAA control mechanisms, however, also affect the chloroplastic pool size due to the rapid transport of IAA between the two compartments.

Indole‐3‐acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

SummaryThe rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β‐glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole‐3‐acetyl)‐β‐D‐glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole‐3‐acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild‐type and transgenic 35S‐rolB tobacco plants and metabolic studies were carried out with [13C6]IAA [2′‐14C]IAA, [14C]IAGluc, [5‐3H]‐2‐o‐(indole‐3‐acetyl)‐myo‐inositol and [14C]indole‐3‐acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.

Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

The rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β-glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole-3-acetyl)-β-D-glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole-3-acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild-type and transgenic 35S-rolB tobacco plants and metabolic studies were carried out with [13C6]IAA [2′-14C]IAA, [14C]IAGluc, [5-3H]-2-o-(indole-3-acetyl)-myo-inositol and [14C]indole-3-acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.

ENDOGENOUS HORMONE LEVELS OF PECAN NUTS DURING STRATIFICATION AND STORAGE AT 10C

An experiment was conducted to determine emergence, free abscisic acid (ABA), and total indoleacetic acid (IAA) levels of 'Hughes' and 'Owens' pecan nuts subjected for 45, 90, and 135 days to stratification and storage at 10C; 'Hughes' nut showed a positive relationship between emergence and days in stratification. However, free ABA and total IAA were not related to days in stratification. Emergence percentages of 'Hughes' and 'Owens' were negatively related to days of storage at 10C. Emergence was not related to levels of free ABA or IAA. Free ABA levels and total IAA levels of 'Hughes' were negatively related to days in storage at 10C.

Leaf age, decline in photosynthesis, and changes in abscisic acid, indole-3-acetic acid, and cytokinin in cotton leaves

Abscisic acid (ABA) decreases stomatal aperture and has been reported to decrease photosynthesis. Conversely, indole-3-acetic acid (IAA) and cytokinins have been reported to increase stomatal aperture and stimulate photosynthesis. Field experiments were conducted in 1990 and 1991 to determine possible correlations of concentrations of ABA, IAA, and the cytokinin, isopentenyladenosine ([9R]iP), with a decline in photosynthesis that occurs in cotton ( Gossypium hirsutum L.) leaves as they age. Sympodial leaves were tagged 31 July 1990 at the first node of fruiting branches when the flower was at anthesis at that node. Photosynthesis was measured at intervals thereafter during the next 37 days as the tagged leaves became older. Mainstem leaves were selected at 4, 8, 12 and 16 nodes below the apex on 7 August 1991. Photosynthesis rates were determined in full sunlight and the leaves then harvested and analyzed for free and conjugated ABA, free and ester IAA, and the cytokinin, [9R]iP. Free ABA decreased, rather than increased, with leaf age. Although conjugated ABA increased with leaf age, it is though to be sequestered in vacuoles and, therefore, metabolically inaccessible. [9R]iP changed little with leaf age. Ester IAA increased with leaf age and was negatively correlated with photosynthesis. Free IAA decreased with leaf age and was significantly correlated with photosynthesis.

The role of the conversion of IBA into IAA on root regeneration in apple: introduction of a test system

A test system for root regeneration was developed that consists of stem slices of apple shoots (ca. 0.5 mm thick; fresh weight ca. 1 mg). Roots regenerated synchronously without intervening callus formation and without interference with compounds originating from other parts of the plant. Supply of indolebutyric acid (IBA) or indoleacetic acid (IAA) induced maximally an average of 8 or 4.5 roots per slice, respectively. After uptake of IBA, a high degree of conjugation resulted in a recovery of 2.5% as internal free IBA (ca. 2 times the medium concentration). Due to conversion of absorbed IBA into IAA a fraction of 0.4% was recovered as (physiologically active) free IAA. After incubation on medium with IAA, 0.5% of the absorbed hormone was recovered in the free acid form. No conversion of IAA into IBA was observed. Equimolar contents of internal free IAA after incubation on IBA or IAA resulted for IBA in a higher number of roots than for IAA. This means that IBA may also act via internal free IBA or may synergistically modify the action or endogenous synthesis of IAA.

Involvement of Abscisic Acid in Ethylene-Induced Cotyledon Abscission in Cotton Seedlings.

Cotton (Gossypium hirsutum L. cv LG102) seedlings raised from seeds exposed to 100 [mu]M norflurazon (NFZ) during imbibition contained reduced levels of free abscisic acid (ABA) and were visibly achlorophyllous. Exposure of untreated cotton seedlings to ethylene concentrations >1 [mu]L/L for 24 h resulted in cotyledon abscission. In contrast, exposure of NFZ-treated seedlings to concentrations of ethylene [less than or equal to]50 [mu]L/L elicited no cotyledon abscission. Application of ABA, an ABA analog, or jasmonic acid to NFZ-treated seedlings restored ethylene-induced abscission. Isolated cotyledonary node explants prepared from NFZ-treated seedlings exhibited an altered dose-response pattern of ethylene-induced petiole abscission. Endogenous levels of free IAA were unaltered in NFZ-treated seedlings. Ethylene treatment (50 [mu]L/L, 24 h) had no effect on free indoleacetic acid (IAA) levels in either control or NFZ-treated seedlings. Levels of conjugated (ester plus amide) IAA were substantially increased in NFZ-treated seedlings regardless of ethylene treatment. These results indicate that endogenous ABA plays an essential, but physiologically undefined, role in ethylene-induced cotyledon abscission in cotton.

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants, and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.

The Phytohormone Profile of the Red Alga Porphyra perforata

Indole-3-acetic acid (IAA) was identified in Porphyra perforata. J. Ag. both as a free auxin and as an auxin conjugate. The quantities of free IAA and IAA released from the conjugates, determined by combined gas chromatography-mass spectrometry (GC-MS), were 20 ng kg −1 FW and 80 ng kg −1 FW respectively. The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), was detected at a level of 22.4 μg kg −1 FW by GC-MS, quantified with deuterated ACC as internal standard. GC-MS analysis of appropriate chromatographic fractions from precalibrated columns failed to detect any abscisic acid (ABA). No evidence was found for the presence of gibberellins

Amide-Linked Indoleacetic Acid Conjugates May Control Levels of Indoleacetic Acid in Germinating Seedlings of Phaseolus vulgaris

We have shown that amide-linked IAA (indole-3-acetic acid) conjugates accumulated to high levels during maturation of bean seeds (K. Bialek and J.D. Cohen [1989] Plant Physiol 91: 775-779). In the present study, we were interested in the fate of these and other IAA conjugates during seed germination. The content of amide-linked conjugates of IAA in cotyledons declined dramatically during the first hours of imbibition. The rate of decline slowed markedly during the period of the resumption of axis growth. The level of amide-linked IAA conjugates in cotyledons remained relatively high after almost 1 week of germination. The decline of IAA conjugates in cotyledons was followed by a steady increase in the content of both free and amide-linked IAA in the embryonic axes. Amide-linked IAA conjugates were also present in the axes cultured on agar after the cotyledons were removed, which suggests that de novo production of these IAA conjugates occurs in the axis of germinating bean seedlings. A comparison of relative amounts of free and conjugated IAA in the axes of intact seedlings and axes cultured on agar showed lower levels of free IAA and higher levels of conjugated IAA in much slower growing isolated axes. These results suggest a more general role for IAA conjugates in the control of seedling growth than simply to serve as a seed storage form of auxin.

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole‐3‐acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography‐mass spectrometry with [13C6]‐IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h.Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N‐1‐naphthylphthalamic acid and 2,3,5‐tri‐iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5‐tri‐iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.

Involvement of rooting factors and free IAA in the rootability of citrus species stem cuttings

Two-year-old trees of cultivar ‘Rangpur’ lime (Citrus limonia Osb.) and of cultivar ‘Oroblanco’, a triploid pummelo-grapefruit hybrid (Citrus grandis Osb. × Citrus paradisi Macf.), which had not reached flowering stage, were grown under greenhouse conditions. Cuttings from the last vigorous vegetative flush were taken from each species for rooting experiments. Callus formation and percentage of rooting were determined after 19 and 36 days. Endogenous indole-3-acetic acid (IAA) content and avocado rooting promoter (ARP)-like activity were determined in leaves and bark (cortex) of the lower end of the cutting on day of excision and 19 days later. Rooting of the easy-to-root ‘Rangpur’ lime reached 77% after 19 days and 100% after 36 days. At those times the difficult-to-root ‘Oroblanco’ did not root at all or reached 12% rooting, respectively. At Day 19 the level of free IAA in the bark of ‘Rangpur’ lime was 3.5 times higher than that at Day 0, and 3.6 times higher than in the ‘Oroblanco’ bark on the same day. ARP was found in both species at excision day and after 19 days using gas-chromatography analysis. The ARP activity on the day of excision was only slightly higher in the leaves and bark of ‘Rangpur’ lime than in ‘Oroblanco’, but after 19 days ARP-like activity rose approximately 45% in the basal bark of ‘Rangpur’ lime with no such increase in its leaves or in the leaves and bark of ‘Oroblanco’. The differences in IAA level and ARP-like activity in the two citrus cultivars appear to be correlated with their ease of rooting, but it is not possible to tell whether the increase in IAA and ARP over the rooting period is the cause or result of root initiation.

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1'-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1'-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using (2)H oxide and 2,4,5,6,7-[(2)H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.

Some Biochemical and Ultrastructural Aspects of Peach Fruit Development

`Redhaven' peach [Prunus persica (L.) Batsch.] fruit growth, expressed as cheek diameter, displayed a double-sigmoid pattern in which four stages were defined (SI, SII, SIII, SIV). Free IAA concentration, as determined by polyclonal antibodies (PcAb) enzyme-linked immunosorbent assay (ELISA), paralleled fruit growth rate, peaking at 30 and 85 days after full bloom (AFB), concurrently with the exponential phases of growth. The highest peroxidase (EC 1.11.1.7) (POD) and IAA oxidase (IAAox) activities occurred during endocarp lignification. The main structural events described were mesocarp cell division within the first 2 weeks AFB and, later, cell enlargement, modifications of the epicarp cells, lignification of the endocarp, differentiation of the chloroplasts, and changes in their starch content. Chemical name used: indole-3-acetic acid (IAA).

Metabolism of [14C]‐indole‐3‐acetic acid by soybean callus and hypocotyl sections

Metabolism of indole‐3‐acetic acid in soybean [Glycine max (L.) Merr.] was investigated with [1‐14C]‐ and [2‐14C]‐indole‐3‐acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [3H]‐IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco (Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [14C]‐indole‐3‐methanol (IM) was three times more efficient than [2‐14C]‐IAA as substrate for producing this metabolite. It was hydrolyzable by β‐glucosidase (EC 3.2.1.21), yielding an indole and D‐glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole‐3‐methanol‐β‐D‐glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole‐3‐methanol‐glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.

The mechanism by which an asymmetric distribution of plant growth hormone is attained.

Zea mays (sweet corn) seedlings attain an asymmetric distribution of the growth hormone indole-3-acetic acid (IAA) within 3 minutes following a gravity stimulus. Both free and esterified IAA (that is total IAA) accumulate to a greater extent in the lower half of the mesocotyl cortex of a horizontally placed seedling than in the upper half. Thus, changes in the ratio of free IAA to ester IAA cannot account for the asymmetric distribution. Our studies demonstrate there is no de novo synthesis of IAA in young seedlings. We conclude that asymmetric IAA distribution is attained by a gravity-induced, potential-regulated gating of the movement of IAA from kernel to shoot and from stele to cortex. As a working theory, which we call the Potential Gating Theory, we propose that perturbation of the plant's bioelectric field, induced by gravity, causes opening and closing of transport channels in the plasmodesmata connecting the vascular stele to the surrounding cortical tissues. This results in asymmetric growth hormone distribution which results in the asymmetric growth characteristics of the gravitropic response.

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

gamma-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms. (gram fresh weight)(-1) free indoleacetic acid (IAA), 150 nanograms. (gram fresh weight)(-1) ester-conjugated IAA, and 10 to 20 micrograms. (gram fresh weight)(-1) amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms. (gram fresh weight)(-1) of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.