Identification and characterization of longevity assurance gene related to stress resistance in Brassica

Brassica is a very important vegetable group worldwide and different stresses are a major concern for these crops. Enhancement of resistance against biotic and abiotic stresses by exploiting stress resistance related genes offers the most efficient approach to address this concern. In this study, a stress resistance related gene was identified from the full-length cDNA library of Brassica rapa cv. Osome, which was determined to be Brassica longevity assurance protein (BrLAP) after sequence analysis. A comparison study of this gene showed a high degree of homology with other stress resistance related longevity assurance genes and was shown to be expressed in all organs during all of the developmental growth stages. In addition, this gene significantly responded after cold, drought and ABA stress treatments in Chinese cabbage. All these data revealed that this gene may be involved in plant resistance against stresses.Keywords: Brassica rapa, longevity assurance gene, gene expression, biotic and abiotic stres

Genome-Wide Analysis of Glycoside Hydrolase Family 1 β-glucosidase Genes in <i>Brassica rapa</i> and Their Potential Role in Pollen Development

Glycoside hydrolase family 1 (GH1) &#946;-glucosidases (BGLUs) are encoded by a large number of genes, and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in model plants (Arabidopsis and rice) and maize, but not in Brassica species, which are important vegetable crops. In this study, we systematically analyzed B. rapa BGLUs (BrBGLUs), and demonstrated the involvement of several genes in pollen development. Sixty-four BrBGLUs were identified in Brassica databases, which were anchored onto 10 chromosomes, with 10 tandem duplications. Phylogenetic analysis revealed that 64 genes were classified into 10 subgroups, and each subgroup had relatively conserved intron/exon structures. Clustering with Arabidopsis BGLUs (AtBGLUs) facilitated the identification of several important subgroups for flavonoid metabolism, the production of glucosinolates, the regulation of abscisic acid (ABA) levels, and other defense-related compounds. At least six BrBGLUs might be involved in pollen development. The expression of BrBGLU10/AtBGLU20, the analysis of co-expressed genes, and the examination of knocked down Arabidopsis plants strongly suggests that BrBGLU10/AtBGLU20 has an indispensable function in pollen development. The results that are obtained from this study may provide valuable information for the further understanding of &#946;-glucosidase function and Brassica breeding, for nutraceuticals-rich Brassica crops

Differential Effects of Herbicidal Compounds on Cytoplasmic Leakages of Green− − − − and White − − − − Maize Leaf Segments

Using maize green -and white -leaf tissues, we have examined the effect of various chemicals on cytoplasmic leakage with respect to the light requirement or chloroplast targeting for their activities. Oxyfluorfen, oxadiazon, diuron, and paraquat, which are known as representative herbicides acting on plant chloroplasts, caused the electrolyte leakage only in the green tissues, whereas 2, 4-dinitrophenol, rose bengal (singlet oxygen producing chemical) and methyl-jasmoante (senescence-stimulating chemical) play a role both in green-and white -tissues. Benzoyl(a) pyrene, generating superoxide radical upon light illumination, functions only in white tissues. Tralkoxydim, metsulfuron-methyl and norflurazon showed no effect in two tested plant samples. In terms of light requirement in electrolyte leakage activity, diuron, oxyfluorfen, oxadiazon, rose bengal, and benzoyl(a) pyrene absolutely require the light for their functions, but other chemicals did not. Based on these results, we could classify into four different response types according to whether chemicals require light or chloroplasts for their action. This classification is likely to be applied to simply and rapidly identify the requirement of light and chloroplasts for the actions of chemicals, thereby it makes easy to characterize many new herbicides that their action mechanisms are unclear, and to elucidate the mode of action of them

SNP in DFR1 Coding Sequence Is Tightly Associated with Anthocyanin Accumulation in Cabbage (B. oleracea var. capitata f. alba) at Low Temperature

Keeping green leaf color at the time of harvest is one of the important traits for breeding of Brassica oleracea var. capitata f. alba, and this trait is related to low anthocyanin contents. To understand the differential accumulation of anthocyanins in cabbage, we selected high anthocyanin accumulators (HAAs) and low anthocyanin accumulator (LAAs) of cabbages and examined the anthocyanin content and the expression of anthocyanin biosynthesis-related genes. Among many genes investigated, BoDFR1 was found to be closely related to anthocyanin accumulation, even under low temperature (LT) conditions. BoDFR1 sequence analyses between HAAs and LAAs revealed that there is a single nucleotide polymorphism (SNP) (1118T/A) in the coding sequence, which substitutes one amino acid from Leu261 to His261; we named BoDFR1 with His261 substitution as BoDFR1v. This amino acid substitution did not affect dihydroflavonol 4-reductase (DFR) activity and substrate specificity, but the polymorphism showed tight association to the BoDFR1 expression, i.e., high level expression of BoDFR1 and low level expression of BoDFR1v under LT conditions. The high levels of BoDFR1 expression were due to the high levels of BoMYB114 and BobHLH expressions combined with low level expression of BoMYBL2, a repressor MYB. On the other hand, low levels of BoDFR1v expression seemed to be related to very low level expressions of BoMYB114 and BobHLH combined with a high level expression of BoMYBL2. It seems that different expression levels of these regulatory genes for MBW (MYB-bHLH-WD40) complex between HAAs and LAAs regulate BoDFR expression and anthocyanin accumulation. Using a single nucleotide polymorphism (SNP) between BoDFR1 and BoDFR1v, molecular markers for PCR and high resolution melt analyses were developed and validated to distinguish between HAAs and LAAs. Combined use of the BoDFR1 SNP marker with other stress markers, such as a cold tolerant marker, will greatly improve cabbage breeding

Development and Application of a PCR-Based Molecular Marker for the Identification of High Temperature Tolerant Cabbage (Brassica oleracea var. capitata) Genotypes

Global warming accelerates the development of high temperature (HT)- and high humidity (HH)-tolerant varieties. This is further facilitated by the identification of HTHH-tolerant genes and the development of molecular markers based on these genes. To identify genes involved in HTHH tolerance in cabbage (Brassica oleracea var. capitata), we performed RNA-seq analysis of two inbred lines, BN1 (HTHH-tolerant) and BN2 (HTHH-susceptible), and selected trehalose 6- phosphate phosphatase I-2 (BoTPPI-2) as one of the HTHH-tolerant-associated genes. We also developed a segregating F2 population from a cross between BN1 and BN2. RNA-seq results showed that BoTPPI-2 transcript levels were high in the HTHH-tolerant inbred line BN1, but not detectable in the HTHH-susceptible inbred line BN2. The expression pattern of BoTPPI-2 was not related to the expression of heat shock-related genes. Soft rot resistance, used as an indicator of HTHH tolerance, was higher in BN1 than in BN2. F2 individuals similar to BN1 with respect to phenotype appeared to be HTHH-tolerant, whereas BN2-types were susceptible to HTHH. Analysis of the genomic DNA revealed the presence of a long terminal repeat (LTR; ca. 4.6 kb) in the ninth intron of the BoTPPI-2_BN2 allele, thereby suppressing its transcription and exhibiting HTHH phenotype. Except for the LTR insertion, the sequence of BoTPPI-2_BN2 was almost identical to that of BoTPPI-2_BN1. On the basis of the LTR and BoTPPI-2 sequences, we developed a molecular marker to identify HTHH-tolerant genotypes and validated its efficiency using F2 individuals, inbred lines, and cultivars from diverse sources. The marker explained the genetic basis of HTHH tolerance in at least 80%, but not 100%, of the cabbage genotypes. Thus, additional markers associated with HTHH tolerance are needed for perfect selection

Natural variation in CIRCADIAN CLOCK ASSOCIATED 1 is associated with flowering time in Brassica rapa

Flowering time is a very important agronomic trait and the development of molecular markers associated with this trait can facilitate crop breeding. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core oscillator component of circadian rhythms that affect metabolic pathways in plants, has been implicated in flowering time control in Brassica species. CCA1 gene sequences from three Brassica rapa inbred lines, showing either early flowering or late flowering phenotypes, were analyzed and a high level of ets were designed and tested using various inbred lines of B. rapa. The usage of InDel marker was further validated by evaluation of flowering time and high resolution melting (HRM) analysis. Both methods, PCR and HRM, validated the use of newly developed marker. Additional sequence analyses of Brassica plants with diploid (AA, BB or CC) and allotetraploid genomes further confirmed a large number of sequence polymorphisms in the CCA1 gene, including insertions/deletions in the fourth intron. Our results demonstrated that sequence variations in CCA1 can be used to develop valuable trait-related molecular markers for Brassica crop breeding.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

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

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 Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories &ldquo;response to heat&rdquo;, &ldquo;heat acclimation&rdquo;, &ldquo;response to light intensity&rdquo;, &ldquo;response to oxidative stress&rdquo;, and &ldquo;response to temperature stimulus&rdquo; were upregulated under warming treatment in both lines, but genes involved in &ldquo;response to auxin stimulus&rdquo; 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. BrPIF4, BrROF2, and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2, and heat shock memory appear to be indispensable for HT adaptation in B. rapa. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa

Genome-Wide Identification of <i>WRKY</i> Genes and Their Response to Cold Stress in <i>Coffea canephora</i>

WRKY transcription factors are known to play roles in diverse stress responses in plants. Low temperatures limit the geographic distribution of Coffea canephora Pierre ex A.Froehner. The WRKYs of C. canephora are still not well characterized, and the response of C. canephora WRKYs (CcWRKYs) under cold stress is still largely unknown. We identified 49 CcWRKYs from the C. canephora genome to gain insight into these mechanisms. These CcWRKYs were divided into three groups that were based on the conserved WRKY domains and zinc-finger structure. Gene expression analysis demonstrated that 14 CcWRKYs were induced during the cold acclimation stage, 17 CcWRKYs were preferentially upregulated by 4 &#176;C treatment, and 12 CcWRKYs were downregulated by cold stress. Subsequently, we carried out a genome-wide analysis to predict 14,513 potential CcWRKY target genes in C. canephora. These isolated genes were involved in multiple biological processes, and most of them could be grouped by the response to stimulus. Among the putative CcWRKY target genes, 235 genes were categorized into response to the cold process, including carbohydrate metabolic, lipid metabolic, and photosynthesis process-related genes. Furthermore, the qRT-PCR and correlation analysis indicated that CcWRKY might control their putative targets that respond to cold stress. These results provide a basis for understanding the molecular mechanism for CcWRKY-mediated cold responses

Molecular analysis of anthocyanin biosynthesis-related genes reveal BoTT8 associated with purple hypocotyl of broccoli (Brassica oleracea var. italica L.)

Broccoli (Brassica oleracea var. italica L.) is a highly nutritious vegetable that typically forms pure green or purple florets. However, green broccoli florets sometimes accumulate slight purplish pigmentation due to environmental factors which reduces the market value. In the present study, we aimed to develop molecular markers to distinguish broccoli genotypes as pure green or purplish floret color at the early seedling stage. Anthocyanins are known to be involved in the purple pigmentation in plants. The purplish broccoli lines were shown to accumulate purple pigmentation in the hypocotyls of very young seedlings; therefore, the expression profiles of the structural and regulatory genes of anthocyanin biosynthesis were analyzed in the hypocotyls using qRT-PCR. BoPAL, BoDFR, BoMYB114, BoTT8, BoMYC1.1, BoMYC1.2, and BoTTG1 were identified as putative candidate genes responsible for the purple hypocotyl color. BoTT8 was much more highly expressed in the purple than green hypocotyls, and was therefore cloned and sequenced from various broccoli lines, revealing SNP and InDel variations between these genotypes. We tested four SNPs (G>A; A>T; G>C; T>G) in the first three exons and a 14-bp InDel (ATATTTATATATAT) in the BoTT8 promoter in 51 broccoli genotypes, and found these genetic variations could distinguish the green lines, purple lines and F1 hybrids. These novel molecular markers could be useful in broccoli breeding programs to develop a true green or purple broccoli cultivar.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author