A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed

Abstract

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4'-O-glycosyl)-sinapoyl),N'-sinapoylspermidine and N,N'-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

Figure 1.

Identification of Sinapoyl Spermidine Derivatives in Arabidopsis Seeds. (A) HPLC profile of methanolic extracts from wild-type Arabidopsis seed. S1 is N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine, and S2 is N,N′-di(sinapoyl)-spermidine. (B) Structures of the two sinapoyl spermidine derivatives identified in Arabidopsis seed. (C) LC/MS/MS fragmentation of compound S1. The possible structures of the major fragments are shown.

Figure 2.

dSpm Insertion in At2g23510 and the Effect of Knockout Mutation on the Accumulation of Sinapoyl Spermidine Derivatives in Arabidopsis Seeds. (A) A schematic model showing the dSpm insertion in line SM_3_38374. Exons are represented by white boxes. The position of insertion (145 bp into the coding region) is indicated. (B) RT-PCR analysis of transcription of At2g23510 in siliques of the wild type (Col-0) and in the At2g23510 homozygous knockout mutant line (SM_3_38374). RT-PCR with primers for elongation factor1α (ef1α) are shown as a control. (C) HPLC profile of the methanolic extracts from siliques of the wild type (Col-0) and At2g23510 homozygous knockout mutant (At2g23510 ko) lines. The two sinapoyl spermidine derivatives, 4′-O-glycosyl- N¹,N⁸-di(sinapoyl)-spermidine (S1) and N¹,N⁸-di(sinapoyl)-spermidine (S2) are indicated. (D) Levels of S1 and S2 in seeds of the wild type (Co-0), SM_3_38374 (At2g23510 ko), and three independent transformants of SM_3_38374 carrying the promoter 35S:At2g23510 construct (At2g23510 ko/AtSDT ox-1, -2, or -3). FW, fresh weight.

Figure 3.

In Vitro HPLC Assay of Recombinant SDT Protein for the Breakdown of Polyamine Conjugates. (A) In vitro breakdown of polyamine conjugates in the absence of SDT. (B) In vitro breakdown of polyamine conjugates in the presence of SDT.

Figure 4.

Accumulation of Disinapoyl Spermidine and Expression of SDT in Arabidopsis during Seed Germination. (A) Expression of SDT in different organs of Arabidopsis. RNA was extracted from different organs of mature plants. qRT-PCR was performed with gene-specific primers, and ef1α was used a constitutive control. The data represent the mean value (±sd) of two independent biological replicates. (B) Siliques harvested at different stages of development. (C) Contents of 4′-O-hexosyl-N¹,N⁸-di(sinapoyl)-spermidine (S1) and N¹,N⁸-di(sinapoyl)-spermidine (S2) in siliques harvested at the different developmental stages indicated in (B). (D) Relative expression levels of SDT in siliques harvested at the different developmental stages indicated in (B). The data represent the mean value (±sd) of two independent biological replicates. (E) GUS staining of seeds in an untransformed wild-type silique. (F) GUS staining (blue color) of seeds carrying the SDT promoter:GUS fusion.

Figure 5.

Expression Patterns of SDT Determined by GUS Staining of SDT Promoter:GUS Lines and qRT-PCR Analyses and Changing Levels of Conjugated Spermidine in Arabidopsis Seedlings during Germination. GUS staining of SDT promoter:GUS (A) and wild-type (B) Arabidopsis plants during germination. Days after germination are shown for each picture. Bars for 1 to 3 d and 4 to 5 d after germination are 0.2 and 1 mm, respectively. (C) Levels of spermidine conjugates in wild-type Arabidopsis seedlings during germination. The data represent the mean value (±sd) of two independent biological replicates. (D) Relative expression levels of SDT during germination determined by qRT-PCR. The data represent the mean value (±sd) of two independent biological replicates.

Figure 6.

Ectopic Expression of SDT Leads to the Accumulation of Disinapoyl Spermidine Derivatives in Arabidopsis Leaves. (A) qRT-PCR analysis of transcript levels of SDT in leaves of wild-type and SDT overexpressing lines (SDT ox-1, -2, and -3). EF1α was used a constitutive control. The data represent the mean value (±sd) of two independent biological replicates. (B) HPLC profiles of methanolic extracts from leaves of the wild type and an SDT overexpressing line. Sinapoyl spermidine derivatives S1 and S2 are indicated.

Figure 7.

Expression and biochemical function of At2g25150 in Arabidopsis. (A) Expression of At2g25150 in different tissues of Arabidopsis determined by qRT-PCR analysis. RNA was extracted from different tissues of mature 6-week-old plants. qRT-PCR was performed with gene-specific primers, and EF1α was used as a constitutive control. The data represent the mean value (±sd) of two independent biological replicates. (B) GUS staining of wild-type root. (C) GUS staining of SCT promoter:GUS transgenic root. Bars = 100 μm. (D) qRT-PCR analysis of transcript levels of SCT (At2g23510) in leaves of wild-type and SCT overexpressing lines(OX-1, -2, and -3). The data represent the mean value (±sd) of two independent biological replicates. (E) HPLC profiles of methanolic extracts from leaves of wild-type and SCT overexpressing lines. The two coumaroyl spermidine derivatives S3 and S4 are shown. (F) Structures of the two coumaroyl spermidine derivatives identified in leaves and roots of SCT-overexpressing Arabidopsis lines. S3, 4′-O-hexosyl-N¹,N⁸-di(coumaroyl)-spermidine, and S4, N¹,N⁸-di(couamroyl)-spermidine. (G) HPLC profiles of methanolic extracts from roots of wild-type and SCT-overexpressing lines. The two coumaroyl spermidine derivatives S3 and S4 are shown.