Expression analysis of genes encoding extracellular leucine-rich repeat proteins in Arabidopsis thaliana.

Leucine-rich repeat (LRR)-containing proteins have been identified in diverse species, including plants. The diverse intracellular and extracellular LRR variants are responsible for numerous biological processes. We analyzed the expression patterns of Arabidopsis thaliana extracellular LRR (AtExLRR) genes, 10 receptor-like proteins, and 4 additional genes expressing the LRR-containing protein by a promoter: ß-glucuronidase (GUS) study. According to in silico expression studies, several AtExLRR genes were expressed in a tissue- or stage-specific and abiotic/hormone stress-responsive manner, indicating their potential participation in specific biological processes. Based on the promoter: GUS assay, AtExLRRs were expressed in different cells and organs. A quantitative real-time PCR investigation revealed that the expressions of AtExLRR3 and AtExLRR9 were distinct under various abiotic stress conditions. This study investigated the potential roles of extracellular LRR proteins in plant growth, development, and response to various abiotic stresses.

Expression analysis of plant intracellular Ras-group related leucine-rich repeat proteins (PIRLs) in Arabidopsis thaliana.

Arabidopsis thaliana contains a family of nine genes known as plant intracellular Ras-group related leucine-rich repeat (LRR) proteins (PIRLs). These are structurally similar to animals and fungal LRR proteins and play important roles in developmental pathways. However, to date, no detailed tissue-specific expression analysis of these PIRLs has been performed. Therefore, in this study, we generated promoter:GUS transgenic plants for the nine A. thaliana PIRL genes and identified their expression patterns in seedlings and floral organs at different developmental stages. Most PIRL members showed expression in the root apical region and in the vascular tissue of primary and lateral roots. Shoot apex-specific expression was recorded for PIRL1 and PIRL8. Furthermore, PIRL1, PIRL3, PIRL5, PIRL6, and PIRL7 showed distinct expression patterns in flowers, especially in pollen and anthers. In addition, co-expression network analysis identified cases where PIRLs were co-expressed with other genes known to have specific functions related to growth and development. Taken together, the tissue-specific expression patterns of PIRL genes improve our understanding of the functions of this gene family in plant growth and development.

Expression analysis of genes encoding malectin-like domain (MLD)- and leucine-rich repeat (LRR)- containing proteins in Arabidopsis thaliana.

Malectin is a maltose-binding endoplasmic reticulum protein conserved in animals. In Arabidopsis thaliana, we identified four genes that encode malectin-like domain (MLD)- and leucine-rich repeat (LRR)-containing proteins (AtMLLRs): two were receptor-like proteins (AtMLLR1 and 2) and the other two were extracellular proteins (AtMLLR3 and 4). The promoter:G3GFP+promoter:GUS assay indicated the organ- and cell-specific expression of the AtMLLR2 and AtMLLR3 genes.Abbreviations: Cmr: chloramphenicol-resistance marker; G3GFP: G3 green fluorescent protein; GUS: ß-glucuronidase; KD: kinase domain; LRR: leucine-rich repeat; MLD: malectin-like domain; RLK: receptor-like kinase; SP: signal peptide; TMD: transmembrane domain; Tnos: nopaline synthase terminator.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.