Cytokinin-Deficient Transgenic Arabidopsis Plants Show Multiple Developmental Alterations Indicating Opposite Functions of Cytokinins in the Regulation of Shoot and Root Meristem Activity

Abstract

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase ( AtCKX ) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5 : GUS ( ␤ -glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter: GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.

Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins

“…4a,b). This phenotype is also consistent with perturbation of cytokinin status, as increase in cytokinin levels normally inhibit root growth and reduce the size of root meristems [22][23][24] . Thus, we speculated that disrupting AtABCG14 may impair root meristem activity.…”

“…1f-h). This affords us anatomical evidence of cytokinin perturbation in the mutant lines, because, as well as regulating root meristems, cytokinins are key regulators of shoot meristem activity, but in an opposite manner 22,26 . Cytokinin deficiencies reportedly diminish the activities of vegetative and floral shoot meristems, including cambium-mediated secondary growth 4,22,26 .…”

“…These morphological alterations are reminiscent of the symptoms of cytokinin deficiency in the cytokinin receptor mutant lines ahk2/ahk3 (ref. 21) and in plants overexpressing Arabidopsis CYTOKININ OXIDASE (AtCKX) 22 , implying that both mutants might have defective cytokinin signalling pathways or responses. Transformation with a genomic DNA sequence comprising the native AtABCG14 promoter, exons and introns completely restored wild-type morphology in these mutant lines ( Supplementary Fig.…”

“…Thus, we speculated that disrupting AtABCG14 may impair root meristem activity. To test this hypothesis, we employed the CYCB1;1::uidA expression cassette, a widely used mitosis reporter 22,25 , to monitor meristem activities in the primary roots of Arabidopsis. When the atabcg14-1 mutant was crossed with a CYCB1;1::uidA transgenic line, b-glucuronidase (GUS) activity was much weaker in the proximal root meristem of homozygous progeny harbouring the CYCB1;1::uidA transgene in an atabcg14-1 mutant background than in CYCB1;1::uidA-expressing progeny with a wild-type background ( Supplementary Fig.…”

Micropropagation of Alibertia edulis Rich

“…BA induced the sprouting in all treatments only in subculture 1 with a peak between 0.1 and 0.5 mg.L -1 , while in the other subcultures its effect was more pronounced only at 0.1 mg.L -1 . The importance of auxin and cytokinin balance has been reported in regulating the apical dominance as well as in a number of morphogenetic events such as shoot formation and branching (Li and Bangerth, 2003;Werner et al, 2003;Nakagawa et al, 2005). Another factor that may influence the morphogenetic events during in vitro culture is a possible interaction between the endogenous concentrations of plant growth regulators and those present in the culture medium (Pinto et al, 1994;Mercier et al, 2003).…”