Effect of Auxins and Associated Metabolic Changes on Cuttings of Hybrid Aspen

In the present study, an attempt was made to induce rooting from single-node cuttings of hybrid aspen (Populus tremula L. ×P. tremuloides Michx.) with different concentrations of IAA, IBA and NAA during rooting. Among the three auxins used, NAA showed more effective induction on rooting as compared to IAA and IBA at the whole level. Thereafter, NAA was used further in experiments for anatomical and biochemical investigation. The results showed that it took 12 days from the differentiation of primordium to the appearance of young adventitious roots with NAA application. It was found that endogenous IAA, ZR and GA3 levels increased, but ABA decreased in cuttings with 0.54 mM NAA treatment. In contrast to the endogenous IAA level, NAA had negative effect on IAA-oxidase (IAAO) activity. Similarly, the decreased peroxidase (POD) activity, consistent with down-regulation of expressed levels of POD1 and POD2, was observed in NAA-treated cuttings. Whereas, NAA resulted in a higher activity in polyphenol oxidase (PPO) compared to the control cuttings. Collectively, the study highlighted that 0.54 mM NAA is efficient on rooting in hybrid aspen, and its effect on metabolic changes during rooting is discussed, which provide valuable information for propagating hybrid aspen.

DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana

Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.

Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.

Physiological and biochemical effects of morphactin IT 3233 on callus and tumour tissues of Nicotiana tabacum L. cultured in vitro. IV. IAA oxidase activity. Oat coleoptile biotest

IAA oxidase activity in callus and tumour tissue of tobacco subjected to the action of morphactin IT 3233 for shorter and longer periods was determined. Control tumour tissue shows an activity higher by about 40 per cent as compared with that of callus tissue. Morphactin applied for a short time (24-h incubation) does not change the activity of the enzyme. When application is prolonged, a considerable enhancement (up to 140%) of the enzyme activity in callus tissue is observed in dependence on the morphactin concentration. In tumour tissues the activity is stimulated by 45 per cent as compared to control. Oat coleoptile elongation growth induced by IAA is limited to 40 per cent when morphactin is added in the concentrations used for tobacco tissue cultures. The possibility of the morphactin action on tissue growth via IAA metabolism is discussed.

No effect of plant growth retarding compounds and growth stimulators on indolo-3-acetic acid oxidase activity in greening cucumber cotyledons

Cotyledons dissected from 5-day-old etiolated cucumber seedlings were incubated in solutions on AMO-1618, B-Nine, CCC and Phosfon D for 48 h in light. In some tests the retardants were applied in mixed solutions with GA₃ or BAP. IAA oxidase was extracted and purified by means of molecular sieving through a bed of Sephadex G-25. The retardants inhibited chlorophyll synthesis by 50 % or more, and had essentially no effect on IAA oxidase activity per cotyledon basis. GA₃ and BAP also had no effect on enzyme activity in spite of a fact that the compounds stimulated growth of the cotyledons. The crude enzyme extract from B-Nine treated cotyledons showed lower IAA oxidase activity in comparison with the water treated control, the effect being due to a longer lag-phase preceding the initiation of IAA oxidation. KNO₃ strikingly stimulated expansional growth of the cotyledons, the effect being correlated with the accelerated chlorophyll accumulation. KNO₃ had no effect on IAA oxidase activity per cotyledon and decreased it per gram fr wt. It is concluded that [1] the growth rate of cucumber cotyledons is not correlated with IAA oxidase activity, and ;[2] the growth retarding compounds do not affect IAA oxidase system is this tissue.

Changes in auxin activity in tumourous and normal tobacco calluses treated with morphactin IT 3233

The addition of morphactin IT 3233 in 1-40 mg/dm³ concentrations to the medium inhibited the growth <i>in vitro</i> of normal and tumourous tobacco calluses. The auxin activity (estimated by the <i>Avena</i> coleoptile straight growth test) of the acid ether extracts from these tissues increased. The activity of zone I (R_f 0.2-0.4, 0.5, solvent system: butanol:water:ammonia 10:10:1) in normal tissues increased more intensively than that of zone II (Rf 0.6-0.8, 0.9). In tumourous tissues, however, these changes were smaller and they concerned merely zone I of auxin activity (R_f 0.0-0.5). It seems that the mechanism of morphactin activity in both kinds of tissue is different. It may be supposed that the excessive accumulation of auxins induces growth inhibition of tissues. A previously found increase in the activity of IAA-oxidase influenced by morphactin might be considered as an adaptation to a higher level of IAA.