Gibberellins (GAs) are plant hormones that regulate many aspects of plant development including seed germination, stem elongation, flower induction, and anther development. Ca²⁺is a ubiquitous second messenger involved in signal transduction of various environmental and developmental stimuli in eukaryotes. In response to diverse internal and external stimuli, cells generate transient increases in cytosolic Ca²⁺([Ca²⁺]_cyt), varying in amplitude, frequency, duration, intracellular location and timing. One physiological response of plant cells to GAs is an increase in [Ca²⁺]_cyt; however, previous studies using fluorescent Ca²⁺indicators suggest that the increase occurs an hour to several hours after GA application, too slow for Ca²⁺to act as a secondary messenger of GA signaling. We reexamined the effects of GAs on an increase in [Ca²⁺]_cytusing the Ca²⁺sensor protein aequorin in Arabidopsis (Arabidopsis thaliana).

Transgenic plants expressing aequorin were administrated with the active GA, GA₄, showing a sharp increase in [Ca²⁺]_cytwithin a few minutes. In contrast, when inactive GA₉-Me or GA₈was administered, no changes in [Ca²⁺]_cytwere observed. GA concentrations of greater than 10^-7M triggered an increase in [Ca²⁺]_cyt, but levels less than 10^-8M did not. This result is consistent with the GA-binding mode of the GA receptor, GA INSENSITIVE DWARF1 (GID1). Taken together, these results suggest that GA triggers a rapid increase in in a physiological concentration-dependent active form-specific manner. The Ca²⁺channel blockers Co²⁺and La³⁺both had an inhibitory effect on the increase in [Ca²⁺]_cyt, in a similar manner to EGTA chelation, suggesting a role of Ca²⁺channels in the GA-induced increase in [Ca²⁺]_cyt. DELLA proteins are negative regulators in GA signaling. GAs trigger DELLA degradation via the ubiquitin-proteasome pathway, thereby promoting plant growth. Arabidopsis contains five DELLAs, GIBBERELLIN-INSENSITIVE (GAI), REPRESSOR OFga1-3(RGA), RGA-LIKE1 (RGL1), RGL2 and RGL3, all of which display partially overlapping yet distinct functions in repressing GA responses. DELLA degradation was previously observed 30 minutes after GA administration. However, an increase in [Ca²⁺]_cytwas observed within 5 minutes of GA treatment, suggesting that it occurred prior to degradation of DELLAs. RGA lacking 17 amino acids in its N-terminus (RGAΔ17) is not degraded even with GA treatment. An active GA-induced increase in [Ca²⁺]_cytwas observed under RGAΔ17 expression. The pentuple mutantdellaPlacks all Arabidopsis DELLA genes. An increase in [Ca²⁺]_cytlevels specific to active GA was observed indellaP. These results suggest that GA induces an increase in [Ca²⁺]_cytindependent of DELLAs. When GA was administered to transgenic Arabidopsis expressing a green fluorescence protein (GFP)-RGA fusion protein under control of the RGA promoter, GFP-RGA fluorescence in the roots disappeared. The Ca²⁺channel blockers Co²⁺and La³⁺had no effect on the disappearance of GFP-RGA fluorescence, although they inhibited the GA-induced increase in [Ca²⁺]_cyt. These results suggest that the GA-induced increase in [Ca²⁺]_cytand the degradation of DELLAs are independent processes. Our findings showing that the GA-induced increase in [Ca²⁺]_cytoccur via a DELLA-independent signaling pathway provide important information on the GA signaling network.

It is proposed that GID1 is the only GA receptor. Our data suggest that the GA-induced increase in [Ca²⁺]_cytdepends on the activation of Ca²⁺channels via the GID1-GA complex in the cytoplasm. Alternatively, the GID1-GA complex could promote the degradation of Ca²⁺channel repressor, allowing derepression of a Ca²⁺channel. Another possibility is that an unidentified accessory GA receptor other than GID1 is involved in the GA-induced increase in [Ca²⁺]_cyt.

Takeshi Ito, Kanako Okada, Jutarou Fukazawa, Yohsuke Takahashi
Department of Biological Science, Graduate School of Science, Hiroshima University, Japan

Publication

Gibberellin Induces an Increase in Cytosolic Ca2+ via a DELLA-Independent Signaling Pathway.
Okada K, Ito T, Fukazawa J, Takahashi Y
Plant Physiol. 2017 Dec