Yoshihiro Mano

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.

The NtAmI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide

Tobacco (Nicotiana tabacum) Bright Yellow‐2 (BY‐2) cells can be grown in medium containing indole‐3‐acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY‐2 cells. Overexpression of the NtAMI1 gene allowed BY‐2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole‐3‐acetic acid, by NtAMI1.

The AMI1 gene family: indole-3-acetamide hydrolase functions in auxin biosynthesis in plants

Novel genes that function in the conversion of indole-3-acetamide (IAM) into indole-3-acetic acid (IAA), which were previously thought to exist only in the bacterial genome, have been isolated from plants. The finding of the AtAMI1 gene in Arabidopsis thaliana and the NtAMI1 gene in Nicotiana tabacum, which encode indole-3-acetamide hydrolase, indicates the existence of a new pathway for auxin biosynthesis in plants. This review summarizes the characteristics of these genes involved in auxin biosynthesis and discusses the possibility of the AMI1 gene family being widely distributed in the plant kingdom. Its evolutionary relationship to bacterial indole-3-acetamide hydrolase, based on phylogenetic analyses, is also discussed.

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxin-autotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

SummaryHorseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborgs) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.

Transformation in Duboisia spp.

The genus Duboisia consists of three species, Duboisia leichhardtii F. Muell, D. myoporoides R. Br., and D. hopwoodii F. Muell. Duboisia species are woody plants and the former two species produce the largest amounts of tropane alkaloids among the Solanaceae, which includes the other alkaloid-producing plants Atropa, Datura, Hyoscyamus and Scopolia. The major alkaloids present in D. leichhardtii and D. myoporoides are scopolamine and hyoscyamine, important compounds that have been used as spasmolytics and anesthetics. The third species, D. hopwoodii, produces mainly nicotine and nornicotine, both of which are nicotine alkaloids normally found in tobacco plants. Both tropane and nicotine alkaloids are synthesized mainly in the roots of these three species.