Yu Jin Jung

Enhanced resistance to bacterial and fungal pathogens by overexpression of a human cathelicidin antimicrobial peptide (hCAP18/LL-37) in Chinese cabbage

The human cathelicidin antimicrobial protein hCAP18, which includes the C-terminal peptide LL-37, is a multifunctional protein. As a possible approach to enhancing the resistance to plant disease, a DNA fragment coding for hCAP18/LL-37 was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein under the control of the cauliflower mosaic virus 35S promoter region. The construct was then introduced into Brassica rapa. LL-37 expression was confirmed in transgenic plants by reverse transcription-polymerase chain reaction and western blot analysis. Transgenic plants exhibited varying levels of resistance to bacterial and fungal pathogens. The average size of disease lesions in the transgenic plants was reduced to less than half of that in wild-type plants. Our results suggest that the antimicrobial LL-37 peptide is involved in wide-spectrum resistance to bacterial and fungal pathogen infection.

BrRZFP1 a Brassica rapa C3HC4-type RING zinc finger protein involved in cold, salt and dehydration stress

C3HC4-type RING zinc finger proteins are known to be essential in the regulation of plant processes, including responses to abiotic stress. Here, we identify, clone and examine the first C3HC4-type RING zinc finger protein (BrRZFP1) from Brassica rapa under stress conditions. Phylogenetic analysis of BrRZFP1 revealed strong sequence similarity to C3HC4-type zinc finger proteins from Arabidopsis that are induced by abiotic stresses. Diverse environmental stresses, including salt and cold, were found to induce BrRZFP1 transcripts greater than eightfold in B.xa0rapa. Additional strong induction was shown of the stress hormone abscisic acid, together suggesting that BrRZFP1 could play a role as a general stress modulator. Similar profiles of induction for each of these stresses was found in both root and shoot tissues, although at much higher levels in roots. Constitutive expression of BrRZFP1 in Nicotiana tabacum was conducted to further analyse how changes in gene expression levels would affect plant stress responses. BrRZFP1 overexpression conferred increased tolerance to cold, salt and dehydration stresses. This was observed in several assays examining growth status throughout development, including increased germination, fresh weight and length of shoots and roots, as well as enhanced chlorophyll retention. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and that changes in its expression level in plants could increase stress tolerance.

Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice

Plants have adapted the ability to respond to various abiotic stresses such as high salinity, osmotic stress, high and low temperatures, and drought in order to survive. Small heat shock proteins (sHsps) play important and extensive roles in plant defenses against abiotic stresses. Herein, we cloned an sHsp gene from the rice, which we named Oshsp16.9 based on the molecular weight of the protein. Real-time PCR analysis showed that expression of the Oshsp16.9 gene was rapidly and strongly induced by stresses including high-salinity (250 mM NaCl), osmotic stress (300 mM mannitol), 100 μM ABA, cold (4°C) and heat (45°C). Subcellular localization assay indicated that Oshsp16.9 was localized specifically in the cytoplasm. In addition, overexpression of Oshsp16.9 in rice conferred tolerance of transgenic plants to salt and drought stress. Taken together, these results suggest that the Oshsp16.9 gene is an important determinant of stress response in plants.

Development and identification of transgenic rice lines with abiotic stress tolerance by using a full-length overexpressor gene hunting system.

The latest report on the draft genome of Brassica rapa sequence has been published. To elucidate the functions of these genes and to efficiently search for agriculturally useful genes, a Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library from Chinese cabbage was introduced into rice via Agrobacterium-mediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs introduced into individual lines was 900~1,200 bp. Basic Local Alignment System Tool (BLAST) analysis of the FL-cDNA genes revealed that 35.5% have unknown function. Most of the randomly selected transgenic rice lines showed overexpression (92%) of these genes relative to the wild-type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedlings were light green). Morphological evaluation of the transgenic rice lines showed minimal phenotypic alteration (12%). Approximately 25.1% and 22% of the plants were significantly ahead in the days to heading and had elevated chlorophyll content, respectively. Other agronomic traits such as filled grains, number of tiller, panicle length, and culm and plant height were relatively less variable among the transgenic lines. These results provide a resource for defining genes that are associated with tolerance in transgenic rice lines.

Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress.

Key messageOverexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses.AbstractGlutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10xa0days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.

Identification of functional SNPs in genes and their effects on plant phenotypes

Single nucleotide polymorphism (SNP) is an abundant form of genetic variation within individuals of species. DNA polymorphism can arise throughout the whole genome at different frequencies in different species. SNP may cause phenotypic diversity among individuals, such as individuals with different color of plants or fruits, fruit size, ripening, flowering time adaptation, quality of crops, grain yields, or tolerance to various abiotic and biotic factors. SNP may result in changes in amino acids in the exon of a gene (asynonymous). SNP can also be silent (present in coding region but synonymous). It may simply occur in the noncoding regions without having any effect. SNP may influence the promoter activity for gene expression and finally produce functional protein through transcription. Therefore, the identification of functional SNP in genes and analysis of their effects on phenotype may lead to better understanding of their impact on gene function for varietal improvement. In this mini-review, we focused on evidences revealing the role of functional SNPs in genes and their phenotypic effects for the purpose of crop improvements.

Astaxanthin production in transgenicArabidopsis with chyB gene encoding β;-carotene Hydroxylase

Oxycarotenoids, produced through the oxidation of carotenoids, play critical roles in plants. This reaction is mediated by a specific enzyme, β;-carotene hydroxylase, which adds hydroxyl groups to the β;-rings of carotenes. To investigate the effect of the β;-carotene hydroxylase gene (Chyb) on oxycarotenoid biosynthesis, we generated transgenicArabidopsis plants that over-expressedChyb under the control of a 35S promoter. Their levels of zeaxanthin and neoxanthin were two- to three-fold greater relative to the WT, while that of violaxanthin, a final product in the xanlthophyll pathway, was 1.3-fold higher than the control. In contrast, the amount of β;-carotene declined as much as 2.4-fold, depending on the particular transgenic line. Interestingly, astaxanthin was produced in the transgenics, but not in the WT. These data suggest that, with the aid of unknown factors in the host, carotenoids could be converted into metabolites in the astaxanthin biosynthetic pathway. Microarray analysis was used lo identify several genes that were consistently up-or down-regulated in transgenic chyB leaves compared with the controls. Here, we also discuss possible modifications in leaf carotenoids, and the importance of these data from a nutritional standpoint.

Targeted genome editing, an alternative tool for trait improvement in horticultural crops

Improving crops through plant breeding, an important approach for sustainable agriculture, has been utilized to increase the yield and quality of foods and other biomaterials for human use. Crops, including cereals, vegetables, ornamental flowers, fruits, and trees, have long been cultivated to produce high-quality products for human consumption. Conventional breeding technologies, such as natural cross-hybridization, mutation induction through physical or chemical mutagenesis, and modern transgenic tools are often used to enhance crop production. However, these breeding methods are sometimes laborious and complicated, especially when attempting to improve desired traits without inducing pleiotropic effects. Recently, targeted genome editing (TGE) technology using engineered nucleases, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) nucleases, has been used to improve the traits of economically important plants. TGE has emerged as a novel plant-breeding tool that represents an alternative approach to classical breeding, but with higher mutagenic efficiency. Here, we briefly describe the basic principles of TGE and the types of engineered nucleases utilized, along with their advantages and disadvantages. We also discuss their potential use to improve the traits of horticultural crops through genome engineering.

Plant microRNAs in molecular breeding

MicroRNAs are small, endogenous, non-coding RNAs found in plants, animals, and in some viruses, which negatively regulate the expression of genes by promoting the degradation of target mRNAs or by translation inhibition. Ever since the discovery of miRNAs, its biology, mechanisms, and functions were extensively studied in the past two decades. Plant and animal miRNAs both regulate target mRNAs, but they differ in scope of complementarity to their target mRNA. Plant microRNAs are known to play essential roles in a wide array of plant development. To date, there are many studies giving evidence that the regulation of miRNA levels can reprogram plant responses to abiotic (physical environment) and biotic stresses (pathogen and herbivore). Most of these studies were first carried out in the model plant Arabidopsis thaliana. Recently, the trend of miRNA research is furthering its role in crop breeding and its evolutionary origin. In this review, we presented the dynamic biogenesis of microRNAs, the diverse functions of miRNAs in plants, and experimental designs used in studying microRNAs in plants, and most importantly, we presented the applications of microRNA-based technology to improve the resistance of crops in abiotic and biotic stresses.

Molecular characterization of the UDP-glucose 4-epimerase (BrUGE) gene family in response to biotic and abiotic stress in Chinese cabbage (Brassica rapa)

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-d-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UGE gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55xa0% of total BrUGE transcripts in most of the tissues examined. All genes showed organ-specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4, and -5)were shown to respond considerably against salt, drought and abscisic acid treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120xa0mM NaCl for 2xa0weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv. oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.