SIZ1 is a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. siz1-2 and siz1-3 T-DNA insertion alleles that caused freezing and chilling sensitivities were complemented genetically by expressing SIZ1, indicating that the SIZ1 is a controller of low temperature adaptation in plants. Cold-induced expression of CBF/DREB1, particularly of CBF3/DREB1A, and of the regulon genes was repressed by siz1. siz1 did not affect expression of ICE1, which encodes a MYC transcription factor that is a controller of CBF3/DREB1A. A K393R substitution in ICE1 [ICE1(K393R)] blocked SIZ1-mediated sumoylation in vitro and in protoplasts identifying the K393 residue as the principal site of SUMO conjugation. SIZ1-dependent sumoylation of ICE1 in protoplasts was moderately induced by cold. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro. ICE1(K393R) expression in wild-type plants repressed cold-induced CBF3/DREB1A expression and increased freezing sensitivity. Furthermore, expression of ICE1(K393R) induced transcript accumulation of MYB15, which encodes a MYB transcription factor that is a negative regulator of CBF/DREB1. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, leading to low temperature tolerance.

Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of AtSIZ1 (At5g60410) cause Arabidopsis to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in siz1 plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity in vitro , and immunoblot analysis revealed that the protein sumoylation profile is impaired in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes AtPT2 , AtPS2 , and AtPS3 were moderate but clearly greater in siz1 seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in siz1 and wild-type seedlings. However, two other Pi starvation-responsive genes, AtIPS1 and AtRNS1 , are induced more slowly in siz1 seedlings by Pi limitation. PHR1, a MYB transcriptional activator of AtIPS1 and AtRNS1 , is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses.

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H ₂ O ₂ -mediated mitogen-activated protein kinase signaling in plants. H ₂ O ₂ stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana ( AtNDPK2 ). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H ₂ O ₂ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H ₂ O ₂ -activated A. thaliana mitogen-activated protein kinases. In the absence of H ₂ O ₂ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro . Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ . Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H ₂ O ₂ -mediated MAPK signaling in plants.

The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2–SOS3 calcium-dependent protein kinase complex up-regulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2–SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2–SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

Hee Jin Park, Woe-Yeon Kim, and Dae-Jin Yun. Mol. Cells 2016;39:447-59. https://doi.org/10.14348/molcells.2016.0083