The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclindependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca 2+ uptake in signaling, the regulation and significance of PAMPinduced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H + )/K + symporter that mediates a high-affinity uptake during K + deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K + efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K + loss. Taken together, our results position ILK1 as a link between plant defense pathways and K + homeostasis.

Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in planta Pi translocation to maintain optimal growth and development. The study of the specificities and biochemical properties of Pht1 transporters will contribute to improving the current understanding of plant phosphorus homeostasis and use-efficiency. In this study, we show through split in vivo interaction methods and in vitro analysis of microsomal root tissues that Arabidopsis thaliana Pht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1(Y312D), Pht1;1(Y312A) and Pht1;1(Y312F), was used to analyse the role of a putative Pi binding residue (Tyr 312) in Pht1;1 transporter oligomerization and function. The homomeric interaction among Pht1;1 proteins was disrupted by mutation of Tyr 312 to Asp, but not to Ala or Phe. In addition, the Pht1;1(Y312D) variant conferred enhanced Pi transport when expressed in yeast cells. In contrast, mutation of Tyr 312 to Ala or Phe did not affect Pht1;1 transport kinetics. Our study demonstrates that modifications to the Pht1;1 higher-order structure affects Pi transport, suggesting that oligomerization may serve as a regulatory mechanism for modulating Pi uptake.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis () () gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. controls endoreplication during trichome development, and mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for function in vivo. Because even highly divergent members of the family are able to replace function in Arabidopsis trichomes, it is likely that the results obtained here for will apply to other members of this plant-specific family of CDK inhibitors.

The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.