Sherry LeClere

Characterization of a Family of IAA-Amino Acid Conjugate Hydrolases from Arabidopsis

The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-l-amino acid conjugates to inhibit Arabidopsis seedling root growth. We have also determined the ability of purified glutathioneS-transferase (GST) fusions of four ArabidopsisIAA-amino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates in vitro, and GST-ILR1, GST-IAR3, and GST-ILL2 have K m values that suggest physiological relevance. In vivo inhibition of root elongation correlates with in vitro hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.

A Family of Auxin-Conjugate Hydrolases That Contributes to Free Indole-3-Acetic Acid Levels during Arabidopsis Germination

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-β-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.

Inputs to the Active Indole-3-Acetic Acid Pool: De Novo Synthesis, Conjugate Hydrolysis, and Indole-3-Butyric Acid b-Oxidation

The phytohormone auxin is important in virtually all aspects of plant growth and development, yet our understanding of auxin homeostasis is far from complete. Plants use several mechanisms to control levels of the active auxin, indole-3-acetic acid (IAA). Plants can synthesize IAA both from tryptophan (Trp-dependent pathways) and from a Trp precursor but bypassing Trp (Trp-independent pathways). Despite progress in identifying enzymes in Trp-dependent IAA biosynthesis, no single IAA biosynthetic pathway is yet defined to the level that all of the relevant genes, enzymes, and intermediates are identified. In addition to de novo synthesis, vascular plants can obtain IAA from the hydrolysis of IAA conjugates. IAA can be conjugated to amino acids, sugars, and peptides; endogenous conjugates that are active in bioassays and hydrolyzed in plants are likely to be important free IAA sources. Conjugation is also used to permanently inactivate excess IAA, and these conjugates may be distinct from the hydrolyzable conjugates. The peroxisomal (3-oxidation of endogenous indole-3-butyric acid (IBA) also can supply plants with IAA, which may account for part of the auxin activity of exogenous IBA. Compartmentalization of enzymes and precursors may contribute to the regulation of auxin metabolism. IAA obtained through de novo synthesis, conjugate hydrolysis, or IBA β-oxidation may have different functions in plant development, and possible roles for the IAA derived from the various pathways are discussed.

Functional Association of Arabidopsis CAX1 and CAX3 Is Required for Normal Growth and Ion Homeostasis

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolar H+/Ca2+ transporter, and the closely related transporter CAX3. We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H+/Ca2+ transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca2+ hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca2+ and also displayed a 22% reduction in vacuolar H+-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca2+ and other ions. These growth defects were partially suppressed by addition of exogenous Mg2+. The double mutant displayed a 42% decrease in vacuolar H+/Ca2+ transport, and a 47% decrease in H+-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO43−, Mn2+, and Zn2+ and decreased Ca2+ and Mg2+ in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.

Sugar Levels Regulate Tryptophan-Dependent Auxin Biosynthesis in Developing Maize Kernels

The maize (Zea mays) Miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer of developing kernels and catalyzes the conversion of sucrose into glucose and fructose. Mutations in Mn1 result in pleiotropic changes, including a reduction in kernel mass and a recently reported decrease in indole-3-acetic acid (IAA) levels throughout kernel development. Here, we show that mn1-1 basal kernel regions (pedicels and basal endosperm transfer layer) accumulate higher levels of sucrose and lower levels of glucose and fructose between 8 and 28 d after pollination when compared with the wild type, whereas upper regions of mn1 accumulate similar or increased concentrations of sugars. To determine the cause of the reduction in IAA accumulation, we investigated transcript levels of several potential IAA biosynthetic enzymes. We demonstrate that reduced IAA levels most closely correspond to reduced transcript levels of ZmYUCCA (ZmYUC), a newly identified homolog of the Arabidopsis (Arabidopsis thaliana) gene YUCCA. We further demonstrate that ZmYUC catalyzes the N-hydroxylation of tryptamine and that sugar levels regulate transcript levels of ZmYUC, both in in vitro-cultured kernels and in a promoter-reporter fusion in Arabidopsis. These results indicate that developing seeds may modulate growth by altering auxin biosynthesis in response to sugar concentrations.

Cowpea Chloroplastic ATP Synthase Is the Source of Multiple Plant Defense Elicitors during Insect Herbivory

In cowpea (Vigna unguiculata), fall armyworm (Spodoptera frugiperda) herbivory and oral secretions (OS) elicit phytohormone production and volatile emission due to inceptin [Vu-In; +ICDINGVCVDA−], a peptide derived from chloroplastic ATP synthase γ-subunit (cATPC) proteins. Elicitor-induced plant volatiles can function as attractants for natural enemies of insect herbivores. We hypothesized that inceptins are gut proteolysis products and that larval OS should contain a mixture of related peptides. In this study, we identified three additional cATPC fragments, namely Vu-GE+In [+GEICDINGVCVDA−], Vu-E+In [+EICDINGVCVDA−], and Vu-In−A [+ICDINGVCVD−]. Leaf bioassays for induced ethylene (E) production demonstrated similar effective concentration50 values of 68, 45, and 87 fmol leaf−1 for Vu-In, Vu-E+In, and Vu-GE+In, respectively; however, Vu-In−A proved inactive. Shortly following ingestion of recombinant proteins harboring cATPC sequences, larval OS revealed similar concentrations of the three elicitors with 80% of the potential inceptin-related peptides recovered. Rapidly shifting peptide ratios over time were consistent with continued proteolysis and preferential stability of inceptin. Likewise, larvae ingesting host plants with inceptin precursors containing an internal trypsin cleavage site rapidly lost OS-based elicitor activity. OS containing inceptin elicited a rapid and sequential induction of defense-related phytohormones jasmonic acid, E, and salicylic acid at 30, 120, and 240 min, respectively, and also the volatile (E)-4,8-dimethyl-1,3,7-nonatriene. Similar to established peptide signals such as systemin and flg22, amino acid substitutions of Vu-In demonstrate an essential role for aspartic acid residues and an unaltered C terminus. In cowpea, insect gut proteolysis following herbivory generates inappropriate fragments of an essential metabolic enzyme enabling plant non-self-recognition.

IAR4, a Gene Required for Auxin Conjugate Sensitivity in Arabidopsis, Encodes a Pyruvate Dehydrogenase E1α Homolog

The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1α-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1α-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.