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The ABC transporter ABCG22 contributes to suberin formation in Arabidopsis seed coats

초록 (요약문)

The hydrophobic suberin layer acts as a physical barrier against biotic and abiotic stresses. ATP-Binding Cassette G proteins (ABCG) transporters are involved in the export of aliphatic and aromatic suberin monomers to extracellular space, but limited information is known about molecular mechanisms underlying ABCG-mediated export of suberin monomers. In this study, I examined the role of a half transporter ABCG22 in the formation of Arabidopsis seed coat suberin. Fluorescent signals from ABCG22pro:eYFP (enhanced yellow fluorescent protein) driven by the ABCG22 promoter were detected in outer integument 1 of seed coats in Arabidopsis. The isolated abcg22 mutant alleles, abcg22-3 and abcg22-4 displayed decreased autofluorescence under UV and increased permeability to tetrazolium salts compared to wild type (WT). The disruption of ABCG22 caused significant changes in total levels of suberin monomers in abcg22-3 and abcg22-4, by 2%-9% decrease compared to WT. The alteration of ABCG22 also led to a decrease in the levels of C18:1 ωhydroxy fatty acids, C18:1 α, ω-dicarboxylic acids, C22 primary alcohols, and C24 fatty acids in both abcg22 mutant seeds. Moreover, germination and seedling establishment of abcg22-3 and abcg22-4 seeds were more sensitive to salt and osmotic stresses than those of WT. In bimolecular fluorescence complementation assay, ABCG22 forms homodimers and heterodimers with ABCG2, ABCG6 or ABCG20. Taken together, ABCG22 contributes to suberin formation in Arabidopsis seed coats.

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초록 (요약문)

소수성 슈베린 층은 생물학적 및 비생물적 스트레스에 대한 물리적 장벽 역할을 한다. ATP-Binding Cassette G 단백질(ABCG) 수송체는 지방족 및 방향족 슈베린 단량체를 세포외 공간으로 내보내는 데 관여하지만 슈베린 단량체의 ABCG 매개 수출의 기본이 되는 분자 메커니즘에 대해서는 제한된 정보가 알려져 있다. 본 연구에서 애기장대 종피 슈베린의 형성에서 half transporter ABCG22 의 역할을 조사했다. ABCG22promoter 에 의해 구동되는 ABCG22pro:eYFP(enhanced yellow fluorescent protein)의 형광 신호는 애기장대의 종피 outer integument 1 에서 검출되었다. Isolated abcg22 돌연변이 대립유전자인 abcg22-3 및 abcg22-4 는 야생형(WT)에 비해 UV 하에서 자가형광이 감소하고 tetrazolium salts 에 대한 투과성이 증가한 것으로 나타났다. ABCG22 의 파괴는 abcg22-3 및 abcg22-4의 슈베린 단량체의 총 수준에 WT 에 비해 2%-9% 감소하여 상당한 변화를 일으켰다. ABCG22 의 파괴는 두 abcg22 돌연변이 종피에서 C18:1 ω-hydroxy fatty acids, C18:1 α, ω-dicarboxylic acids, C22 primary alcohols 및 C24 fatty acids 수준이 감소했다. 또한 abcg22-3 및 abcg22-4 종자의 발아 및 유묘 형성이 WT 보다 염분 및 삼투압 스트레스에 더 민감했다. BiFC 분석에서 ABCG22 는 ABCG2, ABCG6 또는 ABCG20 과 함께 동종이량체 및 이종이량체를 형성한다. 종합하면, ABCG22 는 애기장대 종피의 슈베린 형성에 기여한다.

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목차

Introduction 1
Materials and methods 12
Plant materials and growth conditions 12
RT-PCR and RT-qPCR analysis 13
Constructs design and expression in transgenic plants 13
Identification of the abcg22-3 and abcg22-4 mutants 15
Suberin analysis 16
Seed coat characterization 17
Seed germination and seedling establishment assays 18
Analysis of mucilage and proanthocyanidins 19
Evolutionary tree analysis 19
Bimolecular fluorescence complementation assay 20
Results 21
Isolation of an ABCG22 isoform and ABCG phylogenetic tree 21
Expression patterns of ABCG22 in various Arabidopsis organs 24
Expression of GUS and eYFP driven by the ABCG22 promoter region in Arabidopsis 29
Isolation of T-DNA-inserted abcg22 knockout mutants alleles 33
Seed coat permeability assays of WT, abcg22-3, and abcg22-4 36
Suberin monomer analysis in WT and abcg22 mutant seed coats 42
Germination and seedling establishment of WT and abcg22 mutants seeds to salt and osmotic stress 45
Formation of ABCG22 homodimers and heterodimers between ABCG22 and ABCG2, ABCG6, or ABCG20 49
Discussion 53
References 59

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