Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP/ADP isopentenyltransferases (in Arabidopsis thaliana, AtIPT1, 3, 4–8 ) and tRNA IPTs (in Arabidopsis, AtIPT2 and 9 ). ATP/ADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis -zeatin ( c Z)-type cytokinins are produced possibly by degradation of cis -hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtIPT s, has not yet been described. We isolated null mutants for all members of the ATP/ADP IPT and tRNA IPT gene families in Arabidopsis . Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans -zeatin ( t Z), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of c Z-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl- and cis -hydroxy isopentenyl-tRNA, and c Z-type cytokinins. These results indicate that whereas ATP/ADP IPTs are responsible for the bulk of isopentenyladenine- and t Z-type cytokinin synthesis, tRNA IPTs are required for c Z-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, t Z-, and c Z-type cytokinin production.

One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5′-monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5′-monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.

Significance: Mechanisms of plant seed dormancy evolved to delay germination to a season favorable for seedling growth. Germination timing is an important adaptive early-life history trait which determines plant fitness in natural and agricultural ecosystems. The DELAY OF GERMINATION 1 ( DOG1 ) gene provides natural genetic variation in dormancy, was the first dormancy-specific gene cloned, and encodes a protein of unknown function. We show here that DOG1 controls dormancy of different species by setting the optimal ambient temperature window for germination. This timing is achieved by temperature-dependent alteration of the gibberellin hormone metabolism, which in turn leads to altered expression of genes required for the biomechanical weakening of the coat encasing the embryo. The conserved DOG1-mediated coat-dormancy mechanism controls the timing of seed germination in a temperature-dependent manner.