Genome-wide analysis of the Shaggy-like kinase genes in Chinese cabbage (Brassica rapa ssp. pekinensis)

Xiangshu Dong,Yoonkang Hur 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07

Shaggy-like kinases (SKs), also known as Glycogen synthase kinase 3 (GSK3) proteins, play many important roles in cellular signaling in animals, fungi and amoebae. In particular, SKs participate in key developmental signaling pathways and also regulate the cytoskeleton. SKs -encoding genes are also present in all land plants and in algae, raising questions about possible ancestral functions in eukaryotes. Unlike in animals and Dictyostelium, land plant SKs are encoded by relatively large multi-gene families whose members share high sequence similarity. Along with the studied 10 ASKs (Arabidopsis shaggy-like kinases) indicate that plant SK proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this study, 18 BrSKs are identified from Chinese cabbage, and they are classified into four groups according to the classification of Arabidopsis. The characterization, classification, gene structure and phylogenetic construction of BrSK proteins are performed. Distribution mapping shows that BrSKs are absented in A02 and A10 chromosome. 8 orthologous gene pairs are shared by Chinese cabbage and Arabidopsis. The expression patterns of BrSK genes exhibit differences in five tissues based on RNA-seq data in public data base. Specially, BrSKβ-1 and BrSKβ-2 show floral buds specifically expressed, which indicate that BrSKβ may play a key role during flower or pollen development. We deomonatrated that suppresion of Arabdiopsis orthology of BrSKβ impaired the late pollen in Arabidopsis plants. Taken together, our analyses provided insights into the characterization of the BrSK genes in Chinese cabbage, providing foundation of further functional studies of those genes. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ008118), Rural Development Administration, Republic of Korea]

Comparative transcriptome profiling of freezing stress responsiveness in two contrasting Chinese cabbage genotypes, Chiifu and Kenshin

Dong, Xiangshu,Im, Su-Bin,Lim, Yong-Pyo,Nou, Ill-Sup,Hur, Yoonkang Springer-Verlag 2014 Genes & Genomics Vol.36 No.2

Freezing stress is a major factor affecting plant growth, crop productivity, and the geographical distribution of plants. To identify freezing-responsive genes in Brassica rapa, we analyzed transcriptome profiles of two contrasting inbred lines with different geographic origins, Chiifu and Kenshin, in control and -4 A degrees C-treated leaves. A total of 3,301 genes were differentially expressed between Chiifu and Kenshin upon freezing treatment. Among these, 67 and 1,633 genes were specifically expressed in Chiifu and Kenshin, respectively. An ortholog (BrTPP1) of Arabidopsis trehalose-6-phosphate phosphatase 1 (TPP1) was specifically and highly induced in Chiifu by freezing treatment. However, most cold-responsive genes, including CBF pathway-related genes, showed similar patterns of expression between Chiifu and Kenshin. Many genes involved in stress responses (i.e., to freezing temperatures) were intrinsically and specifically expressed in Chiifu, which is tolerant of freezing temperatures. The results suggest that the CBF pathway is not the main factor conferring freezing tolerance to Chiifu, but genes that are expressed prior to cold acclimation, along with other regulatory genes, may play important roles in freezing tolerance.

Suppression of ASKβ(AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development

Dong, Xiangshu,Nou, Ill-Sup,Yi, Hankuil,Hur, Yoonkang Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.6

Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of $ASK{\beta}$ (AtSK32), $ASK{\beta}$ antisense transgenic plants were generated, revealing the effects of $ASK{\beta}$ down-regulation in Arabidopsis. Suppression of $ASK{\beta}$ expression specifically interfered with pollen development and fertility without altering the plants' vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of $ASK{\beta}$ and its co-expressed genes. In the aborted pollen of $ASK{\beta}$ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate $ASK{\beta}$ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that $ASK{\beta}$ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

ASKβ Is Required for Late Pollen Development in Arabidopsis

Xiangshu Dong,Yoonkang Hur 한국원예학회 2014 한국원예학회 학술발표요지 Vol.2014 No.5

Gene Identification and Molecular Marker Development Related to High-Temperature Tolerance in Cabbage

Myeong-il Mun,Xiangshu Dong,Sangmoo Lee,Seongmin Kim,Yoonkang Hur 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07

To select genes associated with the high-temperature tolerance from Brassica, two transcriptomic analyses have been used: microarray and RNA Seq. Using two contrasting inbred lines of B. rapa, Chiifu and Kenshin, version 3 microarray (135 K microarray) was conducted to RNA samples extracted from series of 45℃-treated leaves and 29 genes were selected for genomic DNA cloning of cabbage. Of 29 genes, 8 genes contain 40 SNPs, 11 SSRs and 23 In-Del markers that distinguish high-temperature tolerant and susceptible cabbages, BN1 and BN2. These 8 genes include a unknown gene, AP2, SMP, FBD, SKP2B, IAA16, HSP21 and OLI2-2. We also selected 16 cabbage genes from RNA Seq analysis using two inbred lines, BN1 and BN2: 5 genes for BN1-high expression, 5 genes for BN1-specific expression, 5 genes for BN2-specific expression, and BoCaMB. Using RNA sequences, genomic DNAs corresponding to 16 genes have been clones and analyzed to find out molecular markers. Markers were further transformed into PCR-based marker and confirmed with additional cabbage genetic lines. We are currently transforming PCR-makers into SNP markers. To examine function of high-temperature tolerant genes, we also transformed 5 genes into Arabidopsis plants. We will describe detailed methods and results in a poster. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ009085), Rural Development Administration, Republic of Korea]

Identification of source‑sink tissues in the leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) by carbohydrate content and transcriptomic analysis

이정여,Xiangshu Dong,최관,송하영,이한결,허윤강 한국유전학회 2020 Genes & Genomics Vol.42 No.1

Background A leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) is composed of a photosynthetic blade and a nonphotosynthetic large midrib; thus each leaf contains both source and sink tissues. This structure suggests that, unlike in other plants, source-sink metabolism is present in a single leaf of Chinese cabbage. Objective This study was designed to identify the transport route of photosynthetic carbon and to determine whether both source and sink tissues were present in a leaf. Methods Plant samples were collected diurnally. Their carbohydrate contents were measured, and a genome-wide transcriptome analysis was performed using the Br300K microarray. Expression profiles of selected genes were validated using qRT-PCR analysis. Results The presence of two contrasting tissues (blade as source and midrib as sink) in a leaf was demonstrated by (1) diurnal distribution patterns of starch and sucrose content; (2) Gene Ontology (GO) enrichment analysis of microarray data; (3) expression profiles of photosynthetic and sucrose biosynthetic genes; and (4) expression patterns of a variety of sugar transporter genes. Conclusion Source and sink tissues were both present in Chinese cabbage leaves, but the midrib functioned as a sink tissue as well as a site exporting to roots and other sink tissues. Function of most genes discriminating between source and sink tissue appeared to be regulated largely at the post-transcriptional level, not at the transcriptional level.

Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa

( Hayong Song ),( Xiangshu Dong ),( Hankuil Yi ),( Ill Sup Nou ),( Yoonkang Hur ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.2

Flowering time is a very important agronomic trait in Brassica crops and regulation of the time is one of major factor in the breeding program. To understand the control of flowering time in Brassica rapa, we have carried out Br300K microarray with two contrasting Brassica inbred lines, Rapid Cycling B. rapa (RCBr) as rapid cycling type and B. rapa ssp. pekinensis inbred line Chiifu as slow flowering phenotype. Reproductive process-related genes were specifically expressed in RCBr, whereas environmental stimuli-responsive genes in Chiifu. Flowering stimulating genes, such as BrFT and BrSOC1, were preferentially expressed in RCBr, while flowering repressing genes, such as BrFLC and BrMAF4, expressed in Chiifu. Several paralogues present in B. rapa, BrFLCs and BrCOLs, were expressed with paralog-specific pattern depending on flowering phenotypes: i.e., BrFLC1 and BrFLC2, major floral repressors, were expressed in Chiifu, BrFLCL/BrFLC5 in RCBr and BrFLC3 in both plants. The expression of several flowering repressing genes was gradually decreased in RCBr growth, but increased in Chiifu growth. However, the expression of genes involved in photoperiodic flowering was no difference between these two plants under LD and SD conditions, indicating photoperiodic pathway is not major factor to distinguish fast vs. slow flowering in B. rapa. The mechanism underlined in the rapid or fast flowering of RCBr would be further elucidated in association with the controlling mechanism of its short life span.

Why F1 hybrid Did Not Show Heterosis with Chlorosis in CMS Breeding System of Chinese Cabbage?

Yoonkang Hur,Xiangshu Dong 한국원예학회 2010 한국원예학회 학술발표요지 Vol.2010 No.5

Genome-Wide Identification and Characterization of Warming-Related Genes in <i>Brassica rapa</i> ssp. <i>pekinensis</i>

Song, Hayoung,Dong, Xiangshu,Yi, Hankuil,Ahn, Ju Young,Yun, Keunho,Song, Myungchul,Han, Ching-Tack,Hur, Yoonkang MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.6

<P>For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two <I>Brassica rapa</I> inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories “response to heat”, “heat acclimation”, “response to light intensity”, “response to oxidative stress”, and “response to temperature stimulus” were upregulated under warming treatment in both lines, but genes involved in “response to auxin stimulus” were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. <I>BrPIF4</I>, <I>BrROF2</I>, and <I>BrMPSR1</I> are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of <I>BrHSFA2</I>, and heat shock memory appear to be indispensable for HT adaptation in <I>B. rapa</I>. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in <I>B. rapa</I>.</P>

Molecular characterization of Arabidopsis thaliana LSH1 and LSH2 genes

Myungjin Lee,Xiangshu Dong,Hayong Song,Ju Yeon Yang,Soyun Kim,YoonkangHur 한국유전학회 2020 Genes & Genomics Vol.42 No.10

Background Arabidopsis thaliana genome encodes ten DUF640 (short for domain of unknown function 640)/ALOG (short for Arabidopsis LSH1 and Oryza G1) proteins, also known as light-dependent short hypocotyl (LSH) proteins. While some of the LSH genes regulate organ boundary determination and shade avoidance response, the function of most of these genes remains largely unknown. Objective In this study, we aimed to characterize the function of AtLSH1 and AtLSH2 in Arabidopsis. Methods We overexpressed AtLSH1 and AtLSH2 (with or without the FLAG tag) in Arabidopsis Col-0 plants under the control of the 35S promoter. We also generated knockout or knockdown lines of these genes by miRNA-induced gene silencing (MIGS). We conducted intensive phenotypic analysis of these transgenic lines, and fnally performed RNA-seq analysis of two AtLSH2 overexpression (OX) lines. Results Although AtLSH1 and AtLSH2 amino acid sequences showed high similarly, AtLSH2-OX lines showed much higher levels of their transcripts than those of AtLSH1-OX lines. Additionally, overexpression of AtLSH1 and AtLSH2 greatly inhibited hypocotyl elongation in a light-independent manner, and reduced both vegetative and reproductive growth. However, knockout or knockdown of both these AtLSH genes did not afect plant phenotype. Gene Ontology (GO) analysis of diferentially expressed genes (DEGs) identifed by RNA-seq revealed enrichment of the GO term ‘response to stimulus’, included phytohormone-responsive genes; however, genes responsible for the abnormal phenotypes of AtLSH2-OX lines could not be identifed. Conclusion Although our data revealed no close association between light and phytohormone signaling components, overexpression of AtLSH1 and AtLSH2 greatly reduced vegetative and reproductive growth of Arabidopsis plants. This property could be used to generate new plants by regulating expression of AtLSH1 and AtLSH2.