Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants

Kim, D. S.; Lee, I. S.; Jang, Cheol Seong; Kang, Si-Yong; Seo, Yong Weon

doi:10.1007/s00299-005-0943-y

Cited by 11 publications

(12 citation statements)

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“…1; Ishihara et al, 2007;Wakasa and Ishihara, 2009). To date, rice mutants insensitive to Trp analogs have been obtained in conventional mutation breeding programs (Wakasa and Widholm, 1987;Lee and Kameya, 1991;Kim et al, 2005). In addition, conventional transgenic approaches such as constitutive overexpression of the Trp-insensitive AS a-subunit have also been tried for the production of high-Trp plants (Tozawa et al, 2001;Wakasa et al, 2006).…”

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“…In addition, conventional transgenic approaches such as constitutive overexpression of the Trp-insensitive AS a-subunit have also been tried for the production of high-Trp plants (Tozawa et al, 2001;Wakasa et al, 2006). Mature seeds of rice plants produced by transgenic approaches accumulated 1,762 to 5,294 nmol/g Trp (Wakasa et al, 2006), while those produced by conventional mutation approaches accumulated 204 to 810 nmol/g Trp (Lee and Kameya, 1991;Kim et al, 2005).…”

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Application of Gene Targeting to Designed Mutation Breeding of High-Tryptophan Rice

et al. 2011

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Site-directed mutagenesis via gene targeting (GT) based on homologous recombination is the ultimate mutation breeding technology because it enables useful information acquired from structural-and computational-based protein engineering to be applied directly to molecular breeding, including metabolic engineering, of crops. Here, we employed this rationale to introduce precise mutations in OASA2-an a-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp) biosynthesis in rice (Oryza sativa)-via GT, with subsequent selection of GT cells using a Trp analog. The expression level of OASA2 in plants homozygous and heterozygous for modified OASA2 was similar to that of nontransformants, suggesting that OASA2 transcription in GT plants was controlled in the same manner as endogenous OASA2, and that GT could lead to a lower risk of gene silencing than in conventional overexpression approaches. Moreover, we showed that enzymatic properties deduced from protein engineering or in vitro analysis could be reproduced in GT plants as evidenced by Trp accumulation levels. Interestingly, mature seeds of homozygous GT plants accumulated Trp levels 230-fold higher than in nontransformants without any apparent morphological or developmental changes. Thus, we have succeeded in producing a novel rice plant of great potential nutritional benefit for both man and livestock that could not have been selected using conventional mutagenesis approaches. Our results demonstrate the effectiveness of directed crop improvement by combining precision mutagenesis via GT with a knowledge of protein engineering.Comparative genomics and structural-and computational-based protein engineering provide copious amounts of useful information that can be applied to crop improvement. In conventional mutation breeding technology using chemical mutagens and ionizing radiation, it remains quite difficult to select mutants harboring target genes modified exactly as required, because mutations occur randomly. Meanwhile, conventional transformation technology based on information from comparative genomics and protein engineering in crops remains a powerful tool in the field of molecular breeding. Agronomical traits such as herbicide tolerance, stress tolerance, and photosynthetic activity have been modified successfully in several crops using conventional transformation technologies that depend on overexpression of modified, highly functional enzymes (Rao, 2008). Moreover, conventional transformation technology is also essential for introducing exogenous genes that do not exist naturally in target plants, e.g. for the production of recombinant proteins. However, mutational approaches are considered better than conventional transformation approaches when introducing directed mutations into endogenous genes to confer novel traits to crops, because conventional transformation technologies cannot eliminate the endogenous targeted gene itself. Thus, a clear goal of current mutation breeding is to modify targeted endogenous genes directly to yield the ...

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Application of Gene Targeting to Designed Mutation Breeding of High-Tryptophan Rice

et al. 2011

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“…Previously, we reported on an accumulation of the OASA2 transcripts in the mutant lines by an altered anthranilate synthase form after 5MT treatment (Kim et al 2005b). Changes in both the level and sensitivity of an anthranilate synthase can contribute to resistance and hence be responsible for the overproduction of tryptophan.…”

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“…Four 5MT-resistant homozygous M 4 lines were obtained from selfpollination of the regenerated 5MT resistant plants. These mutant rice lines were characterized by elevated free amino acid content and altered anthranilate synthase insensitivity to feedback inhibition by tryptophan suggesting that these properties might contribute to 5MT resistance (Kim et al 2005b).…”

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Identification of differentially expressed genes associated with 5-methyltryptophan resistance in rice mutants

Kim¹,

Jang²,

Kim³

et al. 2009

Biologia plant.

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5-Methyltryptophan (5MT), a tryptophan analog, resistant M 4 rice mutants with high free amino acid contents were obtained through in vitro mutagenesis. To evaluate the 5MT resistance mechanism, a cDNA library was constructed by using the leaves and roots of the 5MT resistant mutant plants. Expressed sequenced tags (ESTs) of 1 019 randomly selected clones were analyzed and then assembled 588 unigens. A total of 389 unigenes had significant homologies with known protein sequences at the NCBI database and the remaining 199 unigenes were designated unidentified genes. These unigens were grouped into 13 categories according to their putative functions. Of the 233 randomly selected clones, 25 were identified as differentially expressed genes between 5MT resistant and 5MT sensitive wild type plants. For further study of the differential expression of the genes, expression patterns of 12 genes related to various biological functions were evaluated in response to 5MT treatment in both the resistant plants and sensitive plants. All of the tested 12 genes exhibited higher expression levels in mutant plants than wild type plants under the 5MT inhibition. These expression patterns of the 12 genes suggested that the genes related to 5MT resistance in the rice mutants have a variety of functions, and yield remarkably diverse expression patterns upon 5MT treatment. Many genes that were identified tend to be related to defense and stress responses, suggesting "cross-talking" between biotic/abiotic stresses including the 5MT treatment. Therefore, 5MT resistant mutants might be of value for identifying genes related to plant defenses and stresses.

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“…지금까 지 벼에서 tryptophan 개량에 관한 연구는 돌연변이육 종 program으로 선발한 5-metyltyptophan 저항성 돌연변 이체 (Wakasa and Widholm 1987;Lee and Kameya 1991;Kim et al 2005), AS alpha-subunit에 관련된 유전자를 과 발현시켜 얻어진 형질전환체 (Tozawa et al 2001;Wakasa et al 2006) 등이 알려져 있으며, 이들 식물체는 tryptophan 함량이 대조구에 비해 월등히 증가되었다. 또한 Trp 은 IAA 및 serotonin과 같은 이차대사산물의 변화를 촉진 시킨다 (Radwanski and Last 1995).…”

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Development of high tryptophan GM rice and its transcriptome analysis

et al. 2015

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Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive α subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content. Keywords

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Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants

Cited by 11 publications

References 30 publications

Application of Gene Targeting to Designed Mutation Breeding of High-Tryptophan Rice

Application of Gene Targeting to Designed Mutation Breeding of High-Tryptophan Rice

Identification of differentially expressed genes associated with 5-methyltryptophan resistance in rice mutants

Development of high tryptophan GM rice and its transcriptome analysis

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