It is well established that auxin:cytokinin ratios can be used to control shoot-formation from callus cultures of plant tissues in vitro. This ratio hypothesis has also been extended to other hormone-dependent morphological features and has become a commonly-accepted explanation of how cytokinins and auxins control plant development. We have used transgenic plants overproducing cytokinin or auxin by ectopic expression of the Agrobacterium tumefaciens ipt, or iaaM and iaaH genes to study the interplay between these two hormones and their control of plant development. We have shown that the absolute hormone levels differ in these plants, as well as their ratios. Furthermore, increased IAA-synthesis has been shown to decrease the pool size of cytokinins, while increased cytokinin synthesis reduces IAA-synthesis in planta. We have also crossed series of transgenic auxin- and cytokinin-overproducing tobacco lines, introducing both types of biosynthetic genes into the same plants. The crosses displayed features characteristic of both cytokinin- and auxin- overproduction. Unexpectedly, they also had hormone levels very similar to wild type levels. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits normally associated with auxin and cytokinin overproduction, and observed in the iaa x ipt cross, can not be explained by differences in auxin:cytokinin ratios in individual organs.

Cytokinin biosynthesis pathways

Earlier efforts to elucidate the biosynthetic origin of cytokinins in plants have been inconclusive. The conversion of adenosine-5¥- monophosphate (AMP) and dimethylallyl- pyrophosphate (DMAPP) to the free cytokinins isopentenyl- adenosine- 5*mono- phosphate (iPAMP) and the corresponding nucleoside (iPA) has for many years been accepted as the major pathway of cytokinin biosynthesis in plants with zeatin cytokinins subsequently formed by hydroxy-lation of iPAMP and its derivatives. We have tested the proposed biosynthesis route using transgenic Arabidopsis, expressing the ipt-gene under the control of a glucocorticoid inducible transcription system. We have developed powerful tools for analyzing cytokinins by mass spectrometry, and for quantitative analysis of cytokinin biosynthesis by in-vivo deuterium labeling. We have presented evidence, using these techniques, for an alternative, iPAMP-independent, biosynthetic pathway of zeatin-type cytokinins, operational in both wildtype and ipt-transgenic plants.

Auxin biosynthesis and metabolism
There are at least three IAA-synthesis pathways present in plants. One is the well described tryptophan dependent pathway and one is the recently detected tryptophan independent pathway (Cohen and co-workers). Quantitative analysis of the relative importance of the different IAA biosynthetic routes is technically demanding, since it must be based on indirect analysis due to the unknown nature of the precursors involved. We have investigated IAA-biosynthesis and turnover in protoplasts from wild-type tobacco, and a transgenic IAA-over-producing line, and we were able to show that both pathways occur in this species and, by quantitative turnover studies, demonstrate that the tryptophan independent pathway is the most important one. In a separate study of initial seed-ling growth in Pinus sylvestris, we have also demonstrated that these two pathways are under strict developmental control.

Auxin- a leaf mediated pulse controling lateral root development
We have been able to identify a pulse of auxin originating from the first developed leaves of Arabidopsis thaliana, which reaches the root system 6-7 days after germination. A detailed analysis of the IAA gradient generated by the transport from the sources, young leaves, to the root tip reveals that this gradient is established as a consequence of the IAA pulse and that it occurs at the same time as the massive phase of lateral root development. We have also demonstrated the existence of an acropetal gradient, increasing upwards from the root tip. We used the auxin transport inhibitor naphthylphthalamic acid (NPA) to disrupt this IAA gradient within root apical tissues. This led to increased levels of the hormone, which reduced root length by inhibiting cell elongation. Disruption of the auxin gradient selectively blocked the first asymmetric transverse division within xylem pole peri-cycle cells, thereby abolishing the formation of lateral root primordia. Based on these findings, we have proposed a 3-step model for auxin regulated lateral root formation that highlights the importance of acropetal and basipetal auxin fluxes during this developmental sequence.

 

Ljung K., Bhalerao R.P. & Sandberg G. () The site of auxin biosynthesis and distribution in Arabidopsis thaliana during vegetative growth. Plant Journal  28:1-11

 

Swarup R., Friml J., Marchant A., Ljung K., Sandberg G., Palme K. & Bennett  M. () Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes and  Development  15:.

 

Hertzberg M., Aspeborg H., Schrader J., Andersson A., Blomqvist K., Bhalerao R., Rhaman D., Uhlen M., Teeri T., Lundeberg J., Sundberg B., Nilsson P & Sandberg G () A transcriptional roadmap to wood formation. Proceedings of the National Academy of Science 98:2.

 

Bhalerao R.P., Eklöf J., Ljung K., Marchant A., Bennett M. & Sandberg G. ()  Shoot derived Auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant  Journal 9:325

 

Friml J., Benkova E., Bliou I., Winieska J., Hamann T., Ljung K., Woody S., Sandberg G., Scheres B., Jurgens G. & Palme K. () AtPIN4 mediates sink driven auxin gradients and patterning in Arabidopsis roots. Cell,  108:661.

 

Marchant A., Bhalerao R.P., Casimiro I., Eklöf J., Ljung K., Casero P., Bennett M. & Sandberg G. () AUX1 have multiple function in the movement of IAA that facilitates lateral root branching. Plant Cell, 14:589