Role of tyrosine autophosphorylation and methionine residues in BRI1 function in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs), a group of plant growth hormones, control biomass accumulation and biotic and abiotic stress tolerance, and therefore are highly relevant to agriculture. BRs bind to the BR receptor protein, brassinosteroid insensitive 1 (BRI1), which is classified as a serine/threonine (Ser/Thr) protein kinase. Recently, we reported that BRI1 acts as a dual-specificity kinase both in vitro and in vivo by undergoing autophosphorylation at tyrosine (Tyr) residues. OBJECTIVE: In this study, we characterized the increased leaf growth and early flowering phenotypes of transgenic lines expressing the mutated recombinant protein, BRI1(Y831F)-Flag, compared with those expressing BRI1-Flag. BRI1(Y831F)-Flag transgenic plants showed a reduction in hypocotyl and petiole length compared with BRI1-Flag seedlings. Transcriptome analysis revealed differential expression of flowering time-associated genes (AP1, AP2, AG, FLC, and SMZ) between BRI1(Y831F)-Flag and BRI1-Flag transgenic seedlings. We also performed site-directed mutagenesis of the BRI1 gene, and investigated the effect of methionine (Met) substitution in the extracellular domain (ECD) of BRI1 on plant growth and BR sensitivity by evaluating hypocotyl elongation and root growth inhibition. METHODS: The pBIB-Hyg+-pBR-BRI1-Flag construct(Li et al. 2002) was used as the template for SDM with QuickChange XL Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) to make the SDM mutants. After PCR with SDM kit, add 1 µl of Dpn1 to PCR reaction. Incubate at 37 °C for 2 h to digest parental DNA and then transformed into XL10-gold competent cells. Transcriptome analysis was carried out at the University of Illinois (Urbana-Champaign, Illinois, USA). RNA was prepared and hybridized to the Affymetrix GeneChip Arabidopsis ATH1 Genome Array using the Gene Chip Express Kit (Ambion, Austin, TX, USA). RESULTS: Tyrosine 831 autophosphorylation of BRI1 regulates Arabidopsis flowering time, and mutation of methionine residues in the extracellular domain of BRI1 affects hypocotyl and root length. BRI1(M656Q)-Flag, BRI1(M657Q)-Flag, and BRI1(M661Q)-Flag seedlings were insensitive to the BL treatment and showed no inhibition of root elongation. However, BRI1(M665Q)-Flag and BRI1(M671Q)-Flag seedlings were sensitive to the BL treatment, and exhibited root elongation inhibition. the early flowering phenotype of BRI1(Y831F)-Flag transgenic plants is consistent with the expression levels of key flowering-related genes, including those promoting flowering (AP1, AP2, and AG) and repressing flowering (FLC and SMZ).

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

Obesity is associated with increased risk of Alzheimer's disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In light of this, we developed isoform-specific absolute cDNA standards for absolute quantitative real-time PCR (AQ-PCR), and measured transcript copy numbers for three major APP isoforms (APP770, APP751, and APP695), in SAT from C57BL/6 mice fed either a normal or high-fat diet. Expression of all three major APP isoforms was increased in diet-induced obese mice. Transcript copy numbers of APP770 and APP695 correlated with plasma insulin and CCL2 gene expression. The ratios of APP770 and APP751 to APP695 gradually decreased with aging, and correlated with plasma glucose levels. In addition, APP770 was significantly decreased in thiazolidinedione-treated mice. We describe quantification of APP isoform transcripts by AQ-PCR, which allows for direct comparison of gene copy number across isoforms, between experiments, and across studies conducted by independent research groups, which relative quantitative PCR does not allow. Our results suggest a possible role of differential expression of APP isoforms in the development of obesity-related insulin resistance and adipose tissue inflammation. In addition, it is important to determine if altered ratios of APP isoforms in SAT contribute to higher circulating Aß peptides and increased risk of abnormalities in obesity.