• Journal of Plant Biotechnology
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• v.50
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• pp.190-199
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• 2023

Date palm (Phoenix dactylifera L.: Arecaceae) is a dioecious species where only female trees bear fruits. In their natural state, date palms produce dates once a year. However, in Thailand, some trees were observed to produce dates during the off-season, despite no variations in morphology. The availability of such off-season fruits can significantly increase their market value. Interestingly, most female off-season date palms investigated in this study were obtained through micropropagation. Hence, there is an urgent need for genetic markers to distinguish female offseason flowering plantlets within tissue culture systems. In this study, we aimed to develop random amplification of polymorphic DNA-sequence characterized amplified region (RAPD-SCAR) markers for the identification of female off-season flowering date palms cultivated in Thailand. A total of 160 random decamer primers were employed to screen for specific RAPD markers in off-season flowering male and female populations. Out of these, only one primer, OPN-02, generated distinct genomic DNA patterns in female off-season flowering (FOFdp) individuals compared to female seasonal flowering genotypes. Based on the RAPD-specific sequence, specific SCAR primers denoted as FOFdpF and FOFdpR were developed. These SCAR primers amplified a single 517-bp DNA fragment, predominantly found in off-season flowering populations, with an accuracy rate of 60%. These findings underscore the potential of SCAR marker technology for tracking offseason flowering in date palms. Notably, a BLAST analysis revealed a substantial similarity between the SCAR marker sequence and the transcript variant mRNA from Phoenix dactylifera encoding the SET DOMAIN GROUP 40 protein. In Arabidopsis, this protein is involved in the epigenetic regulation of flowering time. The genetic potential of the off-season flowering traits warrants further elucidation.

• Journal of Mushroom
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• v.10 no.3
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• pp.120-128
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• 2012

Recently sawdust cultivation of Shiitake mushroom (Lentinula edodes) is increasing. It is important to make mycelia to be brown on the substrate surface. This browned surface in sawdust cultivation plays an important role like as artificial bark of the oak log, which protects the other pests and suppresses water evaporation in the substrate. In order to isolate genes which related to brown color formation, differential display method was used. Two cDNA fragments obtained by DD-PCR were 1.2 and 1.6kb and these were expressed in white colored mycelia from L. edodes, but not brown colored mycelia. Partial sequencing of these cDNA fragments showed that the 1.6kb cDNA had 100% identity with the microsatellites gene from Dugenia polichroa. However, the other 1.2kb cDNA fragment had poly T tail on 3' region of partial open reading frame on 5' region. The new primer designed based on the sequence of 1.2kb cDNA was constructed. RT-PCR analysis using the newly designed 0.12kb cDNA specific primer showed that the gene was only expressed in white color mycelia, but not in brown color mycelia. Sequence analysis of 5' region of this 1.2kb cDNA revealed that this gene contained partial open reading frame consisted of 110 amino acid. Homology search using DNASIS database showed that this gene had high sequence homology of 66.7% in DNA level and 69.2 % in amino acid level with dTDP-glucose 4,6-dehydratases gene from Arabidopsis thaliata. The dTDP-glucose 4,6-dehydratases gene was known to be function to have tolerance with oxidation stress. These results strongly suggest that this gene isolated from white mycelia of L. edodes might have a function of repressor against mycelia browning. Therefore I designated this gene as BCR (Brown Color Repressor) gene.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.11-18
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• 2006

Salt stress is one of major environmental factors influencing plant growth and development. To identify salt tolerance determinants in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line for salt stress-sensitive mutant (referred to as ssm1) exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root growth assay. This result suggests that ssm1 mutant is involved in ion homeostasis and osmotic compensation in plant. Molecular cloning of the genomic DNA flanking T-DNA insert of ssm1 mutant was achieved by mutant genomic DNA library screening. T-DNA insertion appeared in the first exon of an open reading frame on F3M18.7, which is the same as AtSYP61. SSM1 is SYP61/OSM1 that is a member of the SNARE superfamily of proteins required for vesicular/target membrane fusions and factor related to abiotic stress.

• Korean Journal of Weed Science
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• v.31 no.2
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• pp.134-145
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• 2011

The objectives of this study were to investigate fitness difference in growth and rice yield in herbicide-transgenic rice overexpressing Myxococcus xanthus and Arabidopsis thaliana protoporphyrinogen oxidase (Protox) genes and non-transgenic rice. We also aimed to determine whether these fitness differences are related to ALA synthesizing capacity, accumulation of terapyrroles, reactive oxygen species, lipid peroxidation, and antioxidative enzymes at different growth stages of rice. Plant height of the transgenic rice overexpressing M. xanthus (MX) and A. thaliana (AP37) Protox genes at 43, 50, and 65 days after transplanting (DAT) was significantly lower than that of WT. Number of tiller of PX as well as MX and AP37 at 50 and 65 DAT was significantly lower than that of WT. At harvest time, culm length and yield of MX, PX and AP37 and rice straw weight of MX and AP37 were significantly low compared with WT. The reduction of yield in MX, PX, and AP37 was caused by spikelets per panicle and 1000 grain weight, ripened grain, spikelets per panicle, 1000 grain weight, and ripened grain, respectively. On the other hand, 135 the reduction of yield in MX, PX, and AP37 was also observed in another yearly variation experiment. The reduction of rice growth in MX, PX, and AP37 was observed in seedling stage as well as growth duration in field. There were no differences in tetrapyrrole intermediate Proto IX, Mg-Proto IX and Mg-Proto IX monomethyl ester, reactive oxygen species ($H_2O_2$ and ${O_2}^-$), MDA, antioxidative enzymes (SOD, CAT, POX, APX, and GR) and chlorophyll between transgenic lines and wild type, indicating that accumulated tetrapyrrole intermediate and other parameters were not related to growth reduction in transgenic rice. However, ALA synthesizing capacity in MX, PX, and AP37 at one day after exposure to light and 52 DAT was significantly lower than that of WT. Further study is required to elucidate the mechanisms underlying the growth and yield difference between transgenic and WT lines.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.245-256
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• 2015

In the agricultural industries, LEDs are used as supplementary, as well as main lighting sources in closed cultivation systems. In cultivation using artificial light sources, various light qualities have been tried to supplement fluorescent lamps to promote plant growth and metabolism. Microarray analysis of Brassica rapa seedlings under blue and fluorescent mixed with blue light conditions identified changes in three genes of the glucosinolate pathway. This attracted attention as functional materials highly expressed 3.6-4.6 fold under latter condition. We selected four more genes of the glucosinolate pathway from the Brassica database and tested their expression changes under fluorescent light mixed with red, green, and blue, respectively. Some genes increased expression under red and blue mixed conditions. The Bra026058, Bra015379, and Bra021429; the orthologous genes of CYP79F1, ST5a, and FMOGS-OX1 in Arabidopsis, are highly expressed in Brassica rapa under fluorescent mixed with blue light conditions. Further, Bra029355, Bra034180, Bra024634, and Bra022448; the orthologous genes of MAM1, AOP3, UGT74B1, and BCAT4 in Arabidopsis, are highly expressed in Brassica rapa under fluorescent mixed with red light conditions. The various light conditions had unique effects on the varieties of Brassica, resulting in differences in glucosinolate synthesis. However, in some varieties, glucosinolate synthesis increased under mixed blue light conditions. These results will help to construct artificial light facilities, which increase functional crops production.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.285-292
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• 2011

In plants, heteroblasty reflects the morphological adaptation during leaf development according to the external environmental condition and affects the final shape and size of organ. Among parameters displaying heteroblasty, leaf index is an important and typical one to represent the shape and size of simple leaves. Leaf index factor is eventually determined by cell proliferation and cell expansion in leaf blades. Although several regulators and their mechanisms controlling the cell division and cell expansion in leaf development have been studied, it does not fully provide a blueprint of organ formation and morphogenesis during environmental changes. To investigate genes and their mechanisms controlling leaf index during leaf development, we carried out molecular-genetic and physiological experiments using an Arabidopsis mutant. In this study, we identified macrophylla (mac) which had enlarged leaves. In detail, the mac mutant showed alteration in leaf index and cell expansion in direction of width and length, resulting in not only modification of leaf shape but also disruption of heteroblasty. Molecular-genetic studies indicated that mac mutant had point mutation in ROTUDIFOLIA3 (ROT3) gene involved in brassinosteroid biosynthesis and was an allele of rot3-1 mutant. We named it mac/rot3-5 mutant. The expression of ROT3 gene was controlled by negative feedback inhibition by the treatment of brassinosteroid hormone, suggesting that ROT3 gene was involved in brassinosteroid biosynthesis. In dark condition, in addition, the expression of ROT3 gene was up-regulated and mac/rot3-5 mutant showed lower response, compare to wild type in petiole elongation. This study suggests that ROT3 gene has an important role in control of leaf index during leaf expansion process for proper environmental adaptation, such as shade avoidance syndrome, via the control of brassinosteroid biosynthesis.

• Journal of Plant Biotechnology
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• v.43 no.3
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• pp.347-358
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• 2016

Brassinosteroid (BR), a plant steroid hormone, plays key roles in numerous growth and developmental processes as well as tolerance to both abiotic and biotic stress. To understand the biological networks involved in BR-mediated signaling pathways and stress tolerance, we performed comparative genome-wide transcriptome analysis of a constitutively activated BR bes1-D mutant with an Agilent Arabidopsis $4{\times}44K$ oligo chip. As a result, we newly identified 1,091 (562 up-regulated and 529 down-regulated) significant differentially expressed genes (DEGs). The combination of GO enrichment and protein network analysis revealed that stress-related processes, such as metabolism, development, abiotic/biotic stress, immunity, and defense, were critically linked to BR signaling pathways. Among the identified gene sets, we confirmed more than a 6-fold up-regulation of NB-ARC and FLS2 in bes1-D plants. However, some genes, including TIR1, TSA1 and OCP3, were down-regulated. Consistently, BR-activated plants showed higher tolerance to drought stress and pathogen infection compared to wild-type controls. In this study, we newly developed a useful, comprehensive method for large-scale identification of critical network and gene sets with global transcriptome analysis using a microarray. This study also showed that gain of function in the bes1-D gene can regulate the adaptive response of plants to various stressful conditions.

• Korean Journal of Weed Science
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• v.30 no.2
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• pp.122-134
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• 2010

We investigated the levels of resistance and accumulation of terapyrroles, reactive oxygen species, lipid peroxidation, and antioxidative enzymes for reasons of growth reduction in herbicide-transgenic rice overexpressing Myxococcus xanthus, Arabidopsis thaliana, and human protoporphyrinogen oxidase (Protox) genes. The transgenic rice overexpressing M. xanthus (MX, MX1, PX), A. thaliana (AP31, AP36, AP37), and human (H45, H48, H49) Protox genes showed 43~65, 41~72 and 17~70-fold more resistance to oxyfluorfen, respectively, than the wild type. Among transgenic rice lines overexpressing Protox genes, several lines showed normal growth compared with the wild type, but several lines showed in reduction of plant height and shoot fresh weight under different light conditions. However, reduction of plant height of AP37 was much higher than other lines for the experimental period. On the other hand, the reduction of plant height and shoot fresh weight in the transgenic rice was higher in high light condition than in low light condition. Enhanced levels of Proto IX were observed in transgenic lines AP31, AP37, and H48 at 7 days after seeding (DAS) and transgenic lines PX, AP37, and H48 at 14 DAS relative to wild type. There were no differences in Mg-Proto IX of transgenic lines except for H41 and H48 and Mg-Proto IX monomethyl ester of transgenic lines except for MX, MX1, and PX. Although accumulation of tetrapyrrole intermediates was observed in transgenic lines, their tetrapyrrole accumulation levels were not enough to inhibit growth of transgenic rice. There were no differences in reactive oxygen species, MDA, ALA synthesizing capacity, and chlorophyll between transgenic lines and wild type indicating that accumulated tetrapyrrole intermediate were apparently not high enough to inhibit growth of transgenic rice. Therefore, the growth reduction in certain transgenic lines may not be caused by a single factor such as Proto IX, but by interaction of many other factors.

• Journal of Life Science
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• v.19 no.8
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• pp.1039-1046
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• 2009

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.153-160
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• 2006

Brassica rape is an important species used as a vegetable, oil, and fodder worldwide. It is related phylogenically to Arabidopsis thaliana, which has already been fully sequenced as a model plant. The 'Multinational Brassica Genome Project (MBGP)'was launched by the international Brassica community with the aim of sequencing the whole genome of B. rapa in 2003 on account of its value and the fact that it has the smallest genome among the diploid Brassica. The genome study was carried out not only to know the structure of genome but also to understand the function and the evolution of the genes comprehensively. There are two mapping populations, over 1,000 molecular markers and a genetic map, 2 BAC libraries, physical map, a 22 cDHA libraries as suitable genomic materials for examining the genome of B. rapa ssp. pekinensis Chinese cabbage. As the first step for whole genome analysis, 220,000 BAC-end sequences of the KBrH and KBrB BAC library are achieved by cooperation of six countries. The results of BAC-end sequence analysis will provide a clue in understanding the structure of the genome of Brassica rapa by analyzing the gene sequence, annotation and abundant repetitive DHA. The second stage involves sequencing of the genetically mapped seed BACs and identifying the overlapping BACs for complete genome sequencing. Currently, the second stage is comprises of process genetic anchoring using communal populations and maps to identify more than 1,000 seed BACs based on a BAC-to-BAC strategy. For the initial sequencing, 629 seed BACs corresponding to the minimum tiling path onto Arabidopsis genome were selected and fully sequenced. These BACs are now anchoring to the genetic map using the development of SSR markers. This information will be useful for identifying near BAC clones with the seed BAC on a genome map. From the BAC sequences, it is revealed that the Brassica rapa genome has extensive triplication of the DNA segment coupled with variable gene losses and rearrangements within the segments. This article introduces the current status and prospective of Korea Brassica Genome Project and the bioinformatics tools possessed in each national team. In the near future, data of the genome will contribute to improving Brassicas for their economic use as well as in understanding the evolutional process.