Arabidopsis CULLIN3 genes regulate primary root growth and patterning by ethylene-dependent and -independent mechanisms.

CULLIN3 (CUL3) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases (called CRL3s) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes. Here, we report that in the model plant Arabidopsis thaliana, CUL3 regulates plant growth and development, not only during embryogenesis but also at post-embryonic stages. First, we show that CUL3 modulates the emission of ethylene, a gaseous plant hormone that is an important growth regulator. A CUL3 hypomorphic mutant accumulates ACS5, the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response. Second, we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway. In particular, we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number. This phenotype is suppressed by ethylene-insensitive or resistant mutations. Finally, we identify a function of CUL3 in distal root patterning, by a mechanism that is independent of ethylene. Thus, our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells.

Regulation of ACS protein stability by cytokinin and brassinosteroid.

A major question in plant biology is how phytohormone pathways interact. Here, we explore the mechanism by which cytokinins and brassinosteroids affect ethylene biosynthesis. Ethylene biosynthesis is regulated in response to a wide variety of endogenous and exogenous signals, including the levels of other phytohormones. Cytokinins act by increasing the stability of a subset of ACC synthases, which catalyze the generally rate-limiting step in ethylene biosynthesis. The induction of ethylene by cytokinin requires the canonical cytokinin two-component response pathway, including histidine kinases, histidine phosphotransfer proteins and response regulators. The cytokinin-induced myc-ACS5 stabilization occurs rapidly (<60 min), consistent with a primary output of this two-component signaling pathway. We examined the mechanism by which another phytohormone, brassinosteroid, elevates ethylene biosynthesis in etiolated seedlings. Similar to cytokinin, brassinosteroid acts post-transcriptionally by increasing the stability of ACS5 protein, and its effects on ACS5 were additive with those of cytokinin. These data suggest that ACS is regulated by phytohormones through regulatory inputs that probably act together to continuously adjust ethylene biosynthesis in various tissues and in response to various environmental conditions.

The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels.

Ethylene biosynthesis is directed by a family of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) that convert S-adenosyl-l-methionine to the immediate precursor ACC. Members of the type-2 ACS subfamily are strongly regulated by proteolysis with various signals stabilizing the proteins to increase ethylene production. In Arabidopsis, this turnover is mediated by the ubiquitin/26 S proteasome system, using a broad complex/tramtrack/bric-a-brac (BTB) E3 assembled with the ETHYLENE OVERPRODUCER 1 (ETO1) BTB protein for target recognition. Here, we show that two Arabidopsis BTB proteins closely related to ETO1, designated ETO1-like (EOL1) and EOL2, also negatively regulate ethylene synthesis via their ability to target ACSs for breakdown. Like ETO1, EOL1 interacts with type-2 ACSs (ACS4, ACS5 and ACS9), but not with type-1 or type-3 ACSs, or with type-2 ACS mutants that stabilize the corresponding proteins in planta. Whereas single and double mutants affecting EOL1 and EOL2 do not show an ethylene-related phenotype, they exaggerate the effects caused by inactivation of ETO1, and further increase ethylene production and the accumulation of ACS5 in eto1 plants. The triple eto1 eol1 eol2 mutant phenotype can be effectively rescued by the ACS inhibitor aminoethoxyvinylglycine, and by silver, which antagonizes ethylene perception. Together with hypocotyl growth assays showing that the sensitivity and response kinetics to ethylene are normal, it appears that ethylene synthesis, but not signaling, is compromised in the triple mutant. Collectively, the data indicate that the Arabidopsis BTB E3s assembled with ETO1, EOL1 and EOL2 work together to negatively regulate ethylene synthesis by directing the degradation of type-2 ACS proteins.