Determining the role of a plant LINC complex in stomatal closure

Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes bridge the nucleoskeleton and cytoskeleton and provide structural support for nuclear movement and positioning, as well as a platform for communication between the cytoplasm and the nucleus. LINC complexes consist of inner nuclear membrane Sad1/UNC-84 (SUN) proteins, which interact with the nuclear lamina; and outer nuclear membrane Klarsicht/ANC-1/Syne Homology (KASH) proteins, which can interact with the cytoskeleton. SUN and KASH proteins interact in the perinuclear space via a SUN domain and KASH peptide tail. Two paralogous Arabidopsis KASH proteins, SINE1 and SINE2, differentially regulate calcium fluctuations and actin reorganization during ABA-induced stomatal closure. The importance of the perinuclear and cytoplasmic interactions of SINE1 and SINE2 during ABA-induced stomatal closure are unknown. In addition, the role of SUN proteins and the nuclear lamina in stomatal closure has not been determined.

Here, we have shown the role of (a) nuclear envelope association, (b) membrane association, and (c) interaction with F-actin in SINE1 and SINE2 function in stomatal closure. Mutant proteins with respective domain deletions were tagged with GFP and placed under native promoters into sine1-1 and sine2-1 mutants, subcellular localizations confirmed, and ABA-induced stomatal closure examined. Surprisingly, SINE1 did not require the ability to co-localize with F-actin to rescue stomatal closure, but did require the connection to the nuclear envelope and LINC complex; whereas all the domains of SINE2 were required for stomatal closure. SUN and nuclear lamina components were also found to be required for stomatal closure by examining ABA-induced stomatal closure in a sun double mutant, sun1-KO sun2-KD, and two nuclear lamina mutants, crwn1 and crwn4. Together this suggests a new role for SUN proteins and the nuclear lamina during stomatal closure, in addition to providing a better understanding of the importance of SINE1 and SINE2 domains during stomatal closure.