Plant growth-promoting factor in the salivary gland of the bug, Lygus disponsi

In the salivary gland of Lygus disponsi, the factor promoting the activity of 3-indoleacetic acid (IAA), or the factor inhibiting the activity of IAA-oxidase, was present. It showed a maximum activity in fraction III of the salivary gland solution, 1·6 times as high as that of the IAA control. The significance of the factor in the salivary gland is discussed with respect to injury (malformation). Auxin was not found in the salivary gland.     

Pectinase and plant growth-promoting factors in the salivary glands of the larva of the bug,Lygus disponsi

Pectinase was detected in the salivary gland of the larvae of Lygus disponsi. The pectinase activity per bug may increase gradually from the first instar larva to the adult. The salivary gland of the third instar larva contained substances indirectly promoting plant growth, i.e. the factor promoting the activity of 3-indoleacetic acid (IAA) or the factor inhibiting the activity of IAA-oxidase, whereas those of the fourth and 5th instar larvae seemed to contain, in addition, auxins which directly promote plant growth. Larvae of all instars had a significant promoting factor only in fraction III of the salivary gland solution. The larvae are as toxic as the adults to sugar beet plants. The presence of pectinase and plant growth-promoting factors in the salivary gland is compared among mirid bugs, and their significance is discussed.     

Morphogenic responses of debudded tobacco plants to gibberellic Acid and indole-3-acetic Acid

Stem applications of indole-3-acetic acid (IAA) or gibberellic acid (GA) did not prevent or alter tumor or teratoma formation in debudded tobacco plants (Nicotiana tabacum L., var. One Sucker). The materials produced intense (in case of GA) and moderate (in case of IAA) stem proliferations when applied to debudded plants but were without effect on intact plants.

The results suggest that debudding-tumors are probably not related to or a result of an auxin or gibberellin deficit and that total debudding has a marked physiological effect on the plant. The altered physiological condition of the debudded plant, indicated by its responses to IAA and GA, may likely be related to tumor and teratoma formation.

     

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with K_D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.     

Exogenous phytohormones and the induction of plant galls by insects

The mechanism of gall induction by insects has remained elusive. Previous studies have met with limited success in attempting to induce galls by injection or application of chemical compounds. To determine whether an exogenous source of phytohormones plays a role in gall induction, we injected cytokinin (CK), auxin (IAA), gibberellic acid (GA), and abscisic acid (ABA) in various concentrations, ratios, and combinations into leaf petioles of Capsicum annuum L. cv. California Wonder (Solanaceae). We found that CK + IAA injections lead to gall-like growth in C. annuum. GA enhanced and ABA inhibited gall growth except in the presence of GA. Isopentenyl adenine (IP) was the most effective type of CK at inducing growth. Our work is consistent with the hypothesis that exogenous CK + IAA produced and supplied by insects leads to gall induction. We hypothesize that insects have obtained the capability to induce galls via acquisition of the biosynthetic pathways to produce IAA and trans-zeatin family CKs through microbial symbiosis or lateral gene transfer.     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

Auxin-Gibberellin Interactions in Pea: Integrating the Old with the New

Recent findings on auxin-gibberellin interactions in pea are reviewed, and related to those from studies conducted in the 1950s and 1960s. It is now clear that in elongating internodes, auxin maintains the level of the bioactive gibberellin, GA₁, by promoting GA₁ biosynthesis and by inhibiting GA₁ deactivation. These effects are mediated by changes in expression of key GA biosynthesis and deactivation genes. In particular, auxin promotes the step GA₂₀ to GA₁, catalyzed by a GA 3-oxidase encoded by Mendel’s LE gene. We have used the traditional system of excised stem segments, in which auxin strongly promotes elongation, to investigate the importance for growth of auxin-induced GA₁. After excision, the level of GA₁ in wild-type (LE) stem segments rapidly drops, but the auxin indole-3-acetic acid (IAA) prevents this decrease. The growth response to IAA was greater in internode segments from LE plants than in segments from the le-1 mutant, in which the step GA₂₀ to GA₁ is impaired. These results indicate that, at least in excised segments, auxin partly promotes elongation by increasing the content of GA₁. We also confirm that excised (light-grown) segments require exogenous auxin in order to respond to GA. On the other hand, decapitated internodes typically respond strongly to GA₁ application, despite being auxin-deficient. Finally, unlike the maintenance of GA₁ content by auxin, other known relationships among the growth-promoting hormones auxin, brassinosteroids, and GA do not appear to involve large changes in hormone level.     

Rapid Effects of IAA on Cell Surface Proteins from Intact Carrot Suspension Culture Cells

Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability.     

Hormonal Relations in the Phototropic Response IV. Light-Induced Changes of Endogenous Auxins in the Coleoptile

Beside indoleacetic acid (IAA), 3 auxins were found by chromatographic resolution of acidic fractions of Avena and Zea coleoptile tips. One of these auxins, designated P, occurred at levels of activity approaching those of IAA. The other 2 auxins, termed F and M, occurred at lower levels of activity. When the auxins of the excised coleoptile tips were isolated immediately after equilateral or unilateral irradiation with blue light at first positive energies, the ratio of IAA to the other auxins increases. This rise is the result of a decrease in P and F, and probably an increase in IAA. Light did not affect materially the total auxin content. It is suggested that P and F might be associated with the basipetal transport inequalities of IAA in phototropism.

P has been partially characterized. Its R_F on chromatograms developed in ammoniacal isopropanol is about 0.65. It is converted to IAA in vitro by heat. The ultraviolet absorption spectrum of chromatographically resolved P also suggests an indolyl complex. P is not readily transported basipetally, and the slope of its relative concentration-response curve (Avena section test) is lower than that of IAA. P does not appear to be any of the chemically characterized native auxins.

     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.     

Invertase and auxin-induced elongation in internodal segments of Phaseolus vulgaris

A close positive correlation was observed between segment elongation and the specific activity of soluble acid invertase in stem segments of P. vulgaris incubated for 21 hr in the presence of IAA or of several synthetic auxins and auxin analogues. Optimum concentrations for the stimulation of growth and invertase activity were similar and varied from 10^?6 M (2,4-D) through 10^?5 M (IAA, IBA, α-NAA, β-NAA) to greater than 10^?4 (IPA, PoAA, trans-cinnamic acid). The weak activity of trans-cinnamic acid, a competitive inhibitor of auxin action, may have resulted from cis-trans isomerization during incubation. The concentration of hexose sugars in the segments fell during incubation in the presence of auxin, the greatest decline in hexose concentration occurring in the presence of compounds exhibiting the greatest stimulation of growth.     

Growth-regulator Interactions in the Growth of the Shoot System of Avena sativa Seedlings: I. THE GROWTH OF THE FIRST INTERNODE

1. Segments, 3.5 mm. long, cut from the first internode of Avenasativa seedlings grown in complete darkness respond to bothauxins and gibberellic acid by accelerated extension. 2. The optimum concentration of indole-3-acetic acid (IAA) is10 p.p.m. and of gibberellic acid (GA) is 0.1 p.p.m. 3. The degree of stimulation relative to the growth of controlsegments is affected by the inclusion in the segement of thenode between the internode and coleoptile. Thus the gibberellineffect is greatly increased while the IAA effect is decreased.The optimal concentrations are not affected by inclusion ofthe node. 4. These results can best be explained in terms of the supplyby the node tissue of an endogenous auxin which is necessaryfor the expression of GA action. 5. Numerous factorial experiments demonstrated that there isno detectable interaction between applied IAA and GA in thepromotion of first-internode extension. This implies that thepostulated endogenous auxin which synergized GAA action in (4)is either an active form of IAA produced only in the node tissueor is a completely different auxin. 6. No synergism of growth-promotive action can be detected betweenGA and the two synthetic auxins I-naphthylacetic acid and 2,4-dichlorophenoxyaceticacid. 7. p-chlorophenoxy-iso-butyric acid (PCIB) anc 2,4,6-trichlorophenoxyaceticacid (2,4,6-T) act as weak auxins and thus antagonize competitivelythe promotive action of GA. 8. The anti-auxin -(I-naphythyl-methyl-sulphide)propionic acid(NMSP) antagonizes competitively the promotive action of bothIAA and GA. 9. The facts under (5)–(8) suggest that auxins and GAare acting at the same growth-promotion centres and may competefor them. 10. Growth inhibitions are induced by high concentrations ofPCIB, 2,4,6-T and NMSP. The inhibitions produced by PCIB and2,4,6-T are both synergized by supra-optimal concentrationsof IAA while that of NMSP is synergized by supra-optimal concentrationsof both IAA and GA. This similarity of the effects of IAA andGA suggests that their inhibition actions also are of a closelysimilar nature.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.     

A mutation affecting the synthesis of 4-chloroindole-3-acetic acid

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana,¹ although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further.² Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis.