Studies on Auxin Protectors X. Protector Levels and Lignification in Sunflower Crown Gall Tissue

The lignification and differentiation of phloem fibers in sunflower stems is inhibited by growing crown gall tumors. Crown gall tumor tissue has previously been shown to contain large quantities of auxin protectors. Since auxin protectors are antioxidants which inhibit peroxidase-catalyzed reactions, and since the formation of lignin is known to involve a peroxidase-catalyzed reaction, an investigation was undertaken to examine the relationship between auxin protectors and lignification in sunflower crown gall tissue. Sunflower crown gall tissue placed into media low in mineral content, rapidly lignifies. In the low mineral media, protectors appear in the medium within an hour or two, implying that endogenously-synthesized protectors rapidly leak out of the tissue. In control media, the tissue neither lignified appreciably, nor did it exhibit an excessive amount of protector release. The addition of Ca²⁺ to the low mineral medium markedly slowed, but did not entirely prevent lignification; similarly Ca²⁺ markedly slowed the release of protector into the low mineral medium. Auxin protectors added to the low mineral medium did not inhibit lignification apparently because, in the medium, the protectors are rapidly oxidized to quinones. The addition of catechol, a substance which mimics protector, also failed to inhibit lignification and also formed a colored compound in the medium suggesting o-qui none formation. In contrast, dithiothreitol, a strong anti-oxidant which upon oxidation does not form a strong oxidant (such as o-quinone), when added to the low mineral medium does inhibit lignification. It is suggested that in the in vitro situation lignification and senescence occurs in low mineral media because the protectors leak out rapidly causing the cell's metabolism to favor peroxidase-catalyzed oxidations including those leading to lignification, while in the in vivo situation the excess protectors produced by crown gall tumor tissue diffuse into surrounding tissue, maintaining a reduced state in such tissues and thereby inhibiting differentiation and lignification. The synthesis of large quantities of protectors by the tumor tissue therefore could account for the anaplasia of the bundle caps observed in sunflower internodes in the vicinity of growing crown gall tumors.     

Studies on Auxin Protectors XII. Auxin Protectors and Polyphenol Oxidase in Developing Coffee Fruit

The fruit of the coffee plant (Coffea arabica) was analyzed for auxin protector content. Ripe coffee berries were separated into pit and pulp, ground in buffer, and assayed for auxin protectors. The extracts were then subjected to gel filtration in order to determine the molecular weight of the protector(s). In the pit, a single protector was found with a molecular weight approaching 5000 daltons, while the pulp contained several auxin protectors, the largest of which appeared to be about 1000 daltons. Chromato-graphic studies of various gel filtration fractions showed that protector activity was always associated with spots which exhibited a light blue fluorescence under UV. The changing patterns during coffee fruit development were also investigated. Auxin protector production, and polyphenol oxidase (E.C. 1.10.3.1), an enzyme related to protector metabolism, were assayed at weekly intervals. In the unripe berry, an auxin protector was found with a molecular weight exceeding 200,000 daltons; as the berry ripened the amount of this protector gradually decreased until almost none was present in the ripe berry and the pattern changed to the pattern described above. Polyphenol oxidase content decreased as the berry ripened. Commercially roasted pits, i.e., coffee “beans”, contained very high levels of protector activity. However, gel filtration studies showed this activity to be associated entirely with low molecular weight compounds.     

Studies on Auxin Protectors: IX. Inactivation of Certain Protectors by Polyphenol Oxidase

Protector-II (Pr-II) of the Japanese morning glory (Pharbitis nil Choisy) was inactivated by exposure to polyphenol oxidase. An unidentified protector in the same molecular weight range obtained from sunflower was also inactivated by this enzyme. Earlier speculations that protectors might be lipoprotein in nature were negated by the fact that neither lipase nor protease inactivated the protectors. The protectors were also not inactivated by incubating with α-amylase, DNase, or RNase. Catechol mimics Pr and is inactivated by polyphenol oxidase. The oxidation of catechol to o-quinone is accompanied by a loss of chromophores that absorb ultraviolet light and the appearance of a reddish brown color. Similarly, when the relatively low molecular weight auxin protectors (Pr-II class) were incubated with polyphenol oxidase, their oxidation was also frequently associated with the formation of brown color, and oxidation with H₂O₂ caused a loss of ultraviolet-absorbing chromophores. The data indicate that auxin protectors contain o-dihydroxyphenolic groups at their active site.     

Distribution of Three Auxin Protector Substances in Seeds and Shoots of the Japanese Morning Glory (Pharbitis nil)

The existence of substances which inhibit the enzymatic destruction of auxin in shoots of the Japanese morning glory (Pharbitis nil Choisy) has been confirmed, as has the fact that these substances are distributed in a gradient diminishing from apex to base in a manner indicating a regulatory role in internode elongation and tissue maturation. In addition to the 2 auxin protector substances reported previously (protectors I and II) which appear to account for most of the inhibition of the enzymatic destruction of auxin in young, elongating stem tissue, a third substance, designated as protector A, has been found to be highly active in seeds, and shoot tips of mature plants: In germinating seeds, no protector I or II activity was observed; in stem tips, no protector II and only slight protector I activity was observed. In contrast, old tissue contained no detectable amounts of protector A, but did contain protectors I and II. Between these extremes along the shoot axis, mixtures of the 3 substances were found. The evidence can be interpreted to mean that protector A is degraded into protectors I and II and perhaps translocated in this form. Gel filtration studies indicate that protector A has a molecular weight exceeding 200,000 gm/mole.     

Studies on Auxin Protectors. IV. The Effect of Manganese on Auxin Protector-I of the Japanese Morning Glory

Auxin protector-I of the Japanese morning glory is inactivated by manganese. Experiments carried out in vitro indicate that in the absence of oxygen the manganic, but not the manganous, ion rapidly inactivates the protector. It is clear from these, and other data described in this report, and the results of other workers, that in the presence of oxygen, manganese accelerates auxin inactivation by means of 2 separate and distinct mechanisms: 1) manganese catalyzes the oxidation of auxin protectors, and 2) following the inactivation of the protectors, or in the absence of protectors, accelerates the oxidation of indoleacetic acid by endogenous peroxidases.     

Studies of Auxin Protectors VIII. Evidence that Auxin Protectors Act as Cellular Poisers

Auxin protectors completely inhibit the peroxidase-catalyzed oxidation of indoleacetic acid (IAA). Presumably only when the protector substance itself has been oxidized, does IAA oxidation begin. Reduced nicotinamide-adenine dinucleotide (NADH) mimics the native auxin protectors: In the presence of NADH, the peroxidase-catalyzed oxidation of IAA does not begin until almost all the NADH has been oxidized. Auxin protectors slow the oxidation of NADH in the presence of the peroxidase complex (enzyme plus manganese). However, in the absence of the peroxidase complex, protectors actually accelerate the spontaneous oxidation of NADH. Protectors can also accelerate the oxidation of the dye 2,6-dichlorophenol-indophenol, especially in the presence of manganese. Protector oxidized by boiling with traces of hydrogen peroxide will act as an electron acceptor in the peroxidase-catalyzed oxidation of NADH. The reversible redox role of auxin protectors implies that they can act as cellular poisers.     

Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols

Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H₂O₂, then to use the H₂O₂ as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H₂O₂ (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates.     

Phytochrome-controlled Nyctinasty in Albizzia julibrissin: IV. Auxin Effects on Leaflet Movement and K Flux

Indole-3-acetic acid, α-naphthylacetic acid, and 2,4-dichlorophenoxyacetic acid (0.001 to 1.0 mm) inhibit the nyctinastic closure of excised Albizzia leaflet pairs; antiauxins and auxin analogs are ineffective, and the auxin effects seem not to be mediated by ethylene. Indoleacetic acid (0.001 to 0.1 mm) also promotes rhythmic opening in the dark, but is ineffective during that phase of rhythmic closure (“leaky phase”) which is insensitive to azide. At these concentrations, all of the indoleacetic acid effects are reversible upon transfer of the tissue to water and are linked to alteration of potassium flux in pulvinule motor cells.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Studies on auxin protector substances, IAA-oxidase and peroxidase in cotyledons of Pharbitis nil

The nature of macromolecular "auxin protector substances" causinglag periods rather than inhibition in the rate of IAA oxidationwas reinvestigated. Three different peaks were separated bySephadex gel filtration; each was then examined by means ofenzymatic (IAA oxidase, peroxidase) and electrophoretic techniquesand correlated with the activities of both enzymes and withzymogram patters. The auxin protector activity of the high molecularweight fractions increased after high temperature treatment.On the basis of experiments involving dialysis and chromatographybefore and after heating, auxin protectors appear to be complexesof macromolecules with small molecules. (Received May 18, 1971; )     

Regulation of Auxin Levels in Coleus blumei by Ethylene

An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth “Scarlet Rainbow” revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-¹⁴C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-¹⁴C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-¹⁴C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene.     

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

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

Studies on Auxin Protectors. V. On the Mechanism of IAA Protection by Protector-I of the Japanese Morning Glory

The mechanism of auxin protection by auxin protector-I (Pr-I) of the Japanese morning glory was studied in vitro. Four lines of evidence indicate that Pr-I acts as a strong reductant which prevents the peroxidase-catalyzed oxidation of IAA: 1) The kinetics of the reaction are best explained on this basis. 2) The Pr-I-induced lag preceding auxin destruction by peroxidase is completely eliminated by a strong oxidant such as H₂O₂ at a concentration which does not appreciably affect the reaction rate. 3) Strong organic reductants mimic the Pr-I-induced lag. And 4) when the reaction rate is altered by varying the concentrations of the reactants, or the temperature, the length of the Pr-I-induced lag varies inversely with the reaction rate.     

ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.     

The masking of peroxidase-catalysed oxidation of IAA in Vigna

Partially purified enzyme preparations of extracts of Vigna seedlings exhibited guaiacol-oxidase activity but not IAA-oxidase activity. However, by ageing the enzyme preparations, or by treating them with H₂O₂, it was possible to unmask IAA-oxidase activity. Gel filtration of Vigna extracts on Sepharose yielded separate peaks for IAA-oxidase, guaiacol-oxidase and auxin protectors. The appearance of a separate IAA-oxidase peak reflected the overlap of peroxidase and protector; the apparent difference in the migration rate of IAA-oxidase and guaiacol-oxidase activity proved to be an artifact. The data imply that previous reports of differences between peroxidase and IAA oxidase need to be reinvestigated to rule out the possible effect of contamination by endogenous, high MW auxin protectors. A rapid method for removing most of the auxin protectors and thereby unmasking IAA-oxidase activity is described.     

Auxin Relations of the Woody Shoot: The Distribution of Diffusible Auxin in Shoots of Apple and Plum Rootstock Varieties

Surveys have been made of diffusible auxin in the stem tissuesof growing shoots of apple and plum rootstock varieties. Usingagar plates as carriers auxin was collected from the lower surfaceof isolated stem sections and assayed by the Avena curvaturemethod. The stool and layer shoots studied grew for severalmonths producing many leaves and reaching considerable lengths.The data provide information on selected internodes and showthe auxin status of the shoot at various times during growth,and the auxin gradients down the stem at these various times.Free auxin content of the shoot apex was consistently less thanthat of the internodes below. In 1946 auxin content declinedthroughout growth with a steady positioning of the auxin peakin the upper shoot. In 1947, following a period of drought,when growth almost ceased, a secondary auxin peak occurred positionedin lower internodes distant from the apex. This seasonal contrastwas reflected in the auxin relations of the individual internode,and was observed both in apple and plum. The nature of the auxindecline below the peak region, and the total disappearance offree auxin from the shoot as growth subsides, is discussed.The reappearance of free auxin in mature internodes, which hasnot been transmitted from the stem apex, implies either derivationfrom a stored state or the ability of the internode to produceits own auxin.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

Development of Erect Leaves in a Modern Maize Hybrid is Associated with Reduced Responsiveness to Auxin and Light of Young Seedlings In Vitro

Modern corn (Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection involves changes in responsiveness to light and auxin.Etiolated seedlings of two older corn hybrids 307 and 3306 elongated significantly more than seedlings of a modern corn hybrid 3394. The level of endogenous auxin and activity of PAT in 307 and 3394 were similar. Hybrid 3394 shows resistance to auxin- and light-induced responses at the seedling, cell and molecular levels. Intact 3394 plants exhibited less responsiveness to the inhibitory effect of R, FR and W, auxin, anti-auxin and inhibitors of PAT. In excised mesocotyl tissue 3394 seedlings also showed essentially low responsiveness to NAA. Cells of 3394 were insensitive to auxin- and light-induced hyperpolarization of the plasma membrane. Expression of ABP4 was much less in 3394 than in 307, and in contrast to 307, it was not upregulated by NAA, R and FR. Preliminary analysis of abp mutants suggests that ABPs may be involved in development of leaf angle in corn.Our results confirm the understanding that auxin interacts with light in the regulation of growth and development of young seedlings and suggest that in corn ABPs may be involved in growth of maize seedlings and development of leaf angle. We hypothesize that ABP4 plays an important role in the auxin- and/or light-induced growth responses. We also hypothesize that in the modern corn hybrid 3394, ABP4 is “mutated,” which may result in the observed 3394 phenotypes, including upright leaves.Key Words: auxin, auxin-binding protein, growth, leaf angle, light, maize