Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation

Abstract

SUMMARYDegradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7-GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7-expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis-zeatin (cZ) and N-glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ-deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone.

CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation

“…For example, in Arabidopsis, the only nontargeted CKX, AtCKX7, is restricted to the cytosol (Galuszka et al, 2007;Köllmer et al, 2014). Similarly, in maize, ZmCKX10 lacked a translocation signal and also localized in the cytosol (Smehilová et al, 2009).…”

Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex

“…As displayed in Figure 1A, inhibiting CK degradation with the specific CKX inhibitor, INCYDE, resulted in slightly elevated levels of the CK bases iP and tZ and the riboside iPR, compounds known to be efficiently degraded by CKXs (Gajdosová et al, 2011;Köllmer et al, 2014). Treatment with adenine ( Figure 1B), a known cytokinin transport antagonist (Bürkle et al, 2003;Cedzich et al, 2008), caused a slight decrease of cZ and tZ bases and increased levels of their respective ribosides.…”

“…In parallel, variations in the CK side chain and stereoisomeric configuration divide isoprenoid CKs into isopentenyladenine (iP), trans-zeatin (tZ), dihydrozeatin, and cis-zeatin (cZ) groups. While iP and tZ groups are considered the most important compounds (Sakakibara, 2006), the functional relevance of cZ-type CKs has been elevated within the last decade (Gajdosová et al, 2011;Köllmer et al, 2014).…”

Plant development regulated by cytokinin sinks

“…In contrast, a variant of CKX2 that lacks the N-terminal signal peptide (∆SP-CKX2) and co-localizes with a cytoplasmic marker ( fig. S7D) did not affect the cytokinin response, and neither did CKX7, which also localizes to the cytoplasm (25) (fig. S7E).…”