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.     

Hormone and seed-specific regulation of pea fruit growth

Growth of young pea (Pisum sativum) fruit (pericarp) requires developing seeds or, in the absence of seeds, treatment with gibberellin (GA) or auxin (4-chloroindole-3-acetic acid). This study examined the role of seeds and hormones in the regulation of cell division and elongation in early pea fruit development. Profiling histone H2A and gamma-tonoplast intrinsic protein (TIP) gene expression during early fruit development identified the relative contributions of cell division and elongation to fruit growth, whereas histological studies identified specific zones of cell division and elongation in exocarp, mesocarp, and endocarp tissues. Molecular and histological studies showed that maximal cell division was from -2 to 2 d after anthesis (DAA) and elongation from 2 to 5 DAA in pea pericarp. Maximal increase in pericarp gamma-TIP message level preceded the maximal rate of fruit growth and, in general, gamma-TIP mRNA level was useful as a qualitative marker for expanding tissue, but not as a quantitative marker for cell expansion. Seed removal resulted in rapid decreases in pericarp growth and in gamma-TIP and histone H2A message levels. In general, GA and 4-chloroindole-3-acetic acid maintained these processes in deseeded pericarp similarly to pericarps with seeds, and both hormones were required to obtain mesocarp cell sizes equivalent to intact fruit. However, GA treatment to deseeded pericarps resulted in elevated levels of gamma-TIP mRNA (6 and 7 DAA) when pericarp growth and cell enlargement were minimal. Our data support the theory that cell division and elongation are developmentally regulated during early pea fruit growth and are maintained by the hormonal interaction of GA and auxin.     

Evidence of 4-Cl-IAA and IAA Bound to Proteins in Pea Fruit and Seeds

The auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally in pea vegetative and fruit tissues (Pisum sativum L.). Previous work has shown that 4-Cl-IAA can substitute for the seeds in the stimulation of pea pericarp growth, whereas IAA is ineffective. Both auxins are found as free acids and as low-molecular-weight conjugates from organic solvent-soluble extracts from pea fruit. Here we present evidence for an additional conjugated auxin species that was not soluble in organic solvent and yielded 4-Cl-IAA and IAA after strong alkaline hydrolysis, suggestive of auxin attachment to pea seed and pericarp proteins. The solvent-insoluble conjugated 4-Cl-IAA in young pericarp was on average 15-fold greater than solvent-soluble 4-Cl-IAA. The solvent-insoluble conjugated IAA was approximately half the levels reported for the solvent-soluble IAA fraction. To identify putative 4-Cl-IAA-bound proteins, polyclonal antibodies were raised to 4-Cl-IAA linked to bovine serum albumin protein (BSA). Immunoblots probed with anti-4-Cl-IAA-BSA antiserum detected three to four unique bands (32–40 kDa) in primarily maternal tissues, and a different set of protein bands were detected in mainly embryonic tissues (ca. 65–74 kDa in mature seed). 4-Cl-IAA and IAA were also identified from protein fractions separated by polyacrylamide gel electrophoresis using GC-MS. These data show that the majority of 4-Cl-IAA, the growth-active auxin in young pea pericarp, and significant levels of IAA are linked to protein fractions. Auxin-proteins may function in regulation of free bioactive 4-Cl-IAA and IAA levels, and/or 4-Cl-IAA or IAA may be targeted to specific proteins post-translationally to modify protein function or stability.     

Seed and 4-chloroindole-3-acetic acid regulation of gibberellin metabolism in pea pericarp.

In this study, we investigated seed and auxin regulation of gibberellin (GA) biosynthesis in pea (Pisum sativum L.) pericarp tissue in situ, specifically the conversion of [14C]GA19 to [14C]GA20. [14C]GA19 metabolism was monitored in pericarp with seeds, deseeded pericarp, and deseeded pericarp treated with 4-chloroindole-3-acetic acid (4-CI-IAA). Pericarp with seeds and deseeded pericarp treated with 4-CI-IAA continued to convert [14C]GA19 to [14C]GA20 throughout the incubation period (2-24 h). However, seed removal resulted in minimal or no accumulation of [14C]GA20 in pericarp tissue. [14C]GA29 was also identified as a product of [14C]GA19 metabolism in pea pericarp. The ratio of [14C]GA29 to [14C]GA20 was significantly higher in deseeded pericarp (with or without exogenous 4-CI-IAA) than in pericarp with seeds. Therefore, conversion of [14C]GA20 to [14C]GA29 may also be seed regulated in pea fruit. These data support the hypothesis that the conversion of GA19 to GA20 in pea pericarp is seed regulated and that the auxin 4-CI-IAA can substitute for the seeds in the stimulation of pericarp growth and the conversion of GA19 to GA20.     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.     

Interaction of 4-chloroindole-3-acetic acid and gibberellins in early pea fruit development

In pea, normal pod (pericarp) growth requires the presence of seeds; and in the absence of seeds, gibberellins (GAs) and/or auxins can stimulate pericarp growth. To further characterize the function of naturally occurring pea GAs and the auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), on pea fruit development, profiles of the biological activities of GA3, GA1, and 4-Cl-IAA on pericarp growth were determined separately and in combination on pollinated deseeded ovaries (split-pericarp assay) and nonpollinated ovaries. Nonpollinated ovaries (pericarps) responded differently to exogenous GAs and 4-Cl-IAA than pollinated deseeded pericarps. In nonpollinated pericarps, both GA3 and 4-Cl-IAA stimulated pericarp growth, but GA3 was significantly more active in stimulating all measured parameters of pericarp growth than 4-Cl-IAA. 4-Cl-IAA, GA1, and GA3 were observed to stimulate pericarp growth similarly in pollinated deseeded pericarps. In addition, the synergistic effect of simultaneous application of 4-Cl-IAA and GAs on pollinated deseeded pericarp growth supports the hypothesis that GAs and 4-Cl-IAA are involved in the growth and development of pollinated ovaries.     

Dynamics of the concentration of IAA and some of its conjugates during the induction of somatic embryogenesis in Coffea canephora

Most of the somatic embryogenesis (SE) process requires the presence, either before or during the embryogenic process, of at least one exogenous auxin. This exogenous auxin induces the presence of endogenous auxins, which appears to be essential for SE induction. We found that during the preincubation period of SE in Coffea canephora, there is an important increase in both free and conjugated indole-3-acetic acid (IAA), as well as indole-3-butyric acid. This increase is accompanied by an increase in the expression of YUCCA (CcYUC), TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (CcTAA1), and GRETCHEN HAGEN 3 (GH3) genes. On the other hand, most of the IAA compounds decreased during the induction of SE. The results presented in this research suggest that a balance between free IAA and its amide conjugates is necessary to allow the expression of SE-related genes.     

Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth

Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.     

Molecular properties of 4-substituted indole-3-acetic acids affecting pea pericarp elongation

Pea (Pisum sativum L.) fruit naturally contain the auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA). However, only 4-Cl-IAA can substitute for the seeds in maintaining pea fruit growth in planta. The importance of the substituent at the 4-position of the indole ring was tested by comparing the molecular properties of 4-X-IAA (X = H, Me, Et, F, or Cl) and their effect on the elongation of pea pericarps in planta. Structure-activity is discussed in terms of structural data derived from X-ray analysis, computed conformations in solution, semiempirical shape and bulk parameters, and experimentally determined lipophilicities and NH-acidities. The size of the 4-substituent, and its lipophilicity are associated with growth promoting activity of pea pericarp, while there was no obvious relationship with electromeric effects.     

New methods to analyse auxin-induced growth II: The swelling reaction of protoplasts – a model system for the analysis of auxin signal transduction?

A method for monitoring the time course of auxin-induced volume changesby protoplasts at a high temporal resolution was developed for Zeamays coleoptile protoplasts. Auxins, like indole-3-acetic acid(IAA), induce a rapid change in volume. Immediately after addition ofthis auxin, a transient shrinkage was observed, followed by a long-termswelling response. This reaction occurred in the same time window as thetypical auxin growth response of intact coleoptiles. Active auxins, like1-naphthalene acetic acid (1-NAA) and 4-chloroindole-3-acetic acid(4-Cl-IAA), caused similar volume changes, whereas the inactive analogue2-naphthalene acetic acid (2-NAA) had no effect. The phytotoxinfusicoccin (FC) induced a rapid swelling response. We conclude that thissingle cell system is very adequate to analyse mechanisms of auxinsignal transduction.     

Synthesis and biological activities of 4-trifluoromethylindole-3-acetic acid: a new fluorinated indole auxin

In our studies on the development of new promoters for the root formation of tree cuttings, 4-trifluoromethylindole-3-acetic acid (4-CF(3)-IAA), a new fluorinated auxin, was synthesized via 4-trifluoromethylindole and 4-trifluoromethylindole-3-acetonitrile by using 2-methyl-3-nitrobenzotrifluoride as the starting material. As a control compound for comparing biological activities, 4-methylindole-3-acetic acid (4-CH(3)-IAA) was also synthesized by using 2,3-dimethylnitrobenzene as the starting material. The biological activities of these compounds were compared by three bioassays with those of indole-3-acetic acid and 4-chloroindole-3-acetic acid (4-Cl-IAA), which, like 4-CF(3)-IAA and 4-CH(3)-IAA, has a substituent at the 4-position of the indole nucleus. 4-CF(3)-IAA showed strong root formation-promoting activity with black gram cuttings which was 1.5 times higher than that of 4-(3-indole)butyric acid at 1x10(-4) M. 4-CH(3)-IAA, however, only weakly promoted root formation in spite of its strong inhibition of hypocotyl growth in Chinese cabbage and promotion of hypocotyl swelling and lateral root formation in black gram. On the other hand, 4-CF(3)-IAA demonstrated weaker activities than 4-CH(3)-IAA and 4-Cl-IAA in these two bioassays.     

In vitro binding affinities of 4-chloro-, 2-methyl-, 4-methyl-, and 4-ethylindoleacetic acid to auxin-binding protein 1 (ABP1) correlate with their growth-stimulating activities

4-Chlorindole-3-acetic acid (4-CI-IAA), an endogenous auxin in certain plant species of Fabaceae, has a higher efficiency in stimulating cell elongation of grass coleoptiles compared with indole-3-acetic acid (IAA), particularly at low concentrations. However, some investigations reported a 1,000-fold discrepancy between growth stimulation and binding affinity of 4-CI-IAA to auxin-binding protein 1 (ABP1) from maize. Here we report binding data of 4-CI-IAA and three alkylated IAA derivatives using purified ABP1 in equilibrium dialysis. There is a clear correlation between the growth-promoting effects and the binding affinity to ABP1 of the different IAA analogues measured by competition of [³H]naphthalene-1-acetic acid binding. Our data are consistent with the hypothesis that ABP1 mediates auxin-induced cell elongation.Abbreviations ABP1 auxin-binding protein 1 - 4-CI-IAA 4-chloroindole-3-acetic acid - NAA naphthalene-1-acetic acid - ER endoplasmic reticulum - IAA indole-3-acetic acid - 2-Me-IAA 2-methylindole-3-acetic acid - 4-Me-IAA 4-methylindole-3-acetic acid - 4-Et-IAA 4-ethylindole-3-acetic acid - MES 4-morpholineethanesulfonic acid - PAA phenylacetic acid     

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca²⁺-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca²⁺ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca²⁺ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca²⁺ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS–YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca²⁺ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

One-sentence summary: The immunity-related Ca²⁺ channel CYCLIC NUCLEOTIDE-GATED CHANNEL 2 modulates auxin homeostasis and balances cellular auxin perception by influencing auxin biosynthesis.     

Rooting, growth and ethylene evolution of pea cuttings in response to chloroindole auxins

In pea cuttings ( Pisum sativum L. cv. Alaska) we measured shoot and root growth and ethylene production in response to 4-chloroindole-3-acetic acid (4-CI-IAA) or 4,6-dichloroindole-3-acetic acid (4,6-Cl₂-IAA). Leafy cuttings treated basally with either of the chlorinated auxins in high concentrations showed permanent epinasty, loss of apical growth and dominance resulting in the outgrowth of laterals from the lower-most axillary bud. The naturally occurring 4-CI-IAA was a better root promoter than the synthetic 4,6-Cl₂-IAA which inhibited rooting. Both chloroindole auxins induced very high ethylene evolution, which lasted much longer than the ethylene evolution after IAA treatment.     

Brassinolide-induced elongation and auxin

Segments from the hook and subhook zone of the stem of 6-day-old etiolated Pisum sativum L. cv. Victory Freezer seedlings were used to study the relationship between brassinolide and auxin in the promotion of elongation. Minor changes in exogenous indole-3-acetic acid or4-chloroindole-3-acetic acid concentration affected the kinetics markedly and the ethylene generator ethephon overcame brassinolide-induced elongation in an antagonistic interaction. Brassinolide-induced elongation was markedly inhibited by low concentrations of the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile, and diagnostic concentrations of the antiauxin 2-( p -chlorophenoxy) isobutyric acid did not affect brassinolide-induced elongation. As the characteristics of auxin-induced growth are not displayed in brassinolide-induced elongation of the upper stem segment, it is proposed that brassinolide does not depend on auxin as a mediator in the promotion of elongation of younger tissues but that it can interact in a very complex manner with auxin. In the elongation of more mature tissues, and in bending responses, brassinolide probably accelerates auxin effects. When split, the upper stem segment was unusual in its lack of specific response to growth regulators, and the slight relief of epidermal tension.