Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal

Abstract

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that have an important role in plant development and adaptation to environmental changes. In this study, we identified and characterized a spermidine hydroxycinnamoyl transferase (SHT) from eggplant (Solanum melongena) and its wild relative S. richardii, designated as SmSHT and SrSHT, respectively. SmSHT was abundant in flowers and fruits, whereas the level of SrSHT was remarkably low in all tissues. Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits, indicating its involvement in plant stress response. Both SHT polypeptides had extremely high identity with just five amino-acid substitutions. Recombinant SmSHT catalyzed the synthesis of mono-, bi- and tri- acylated polyamines. Using caffeoyl-CoA as the acyl donor, SmSHT preferred spermidine as the acyl acceptor. When spermidine was the acyl acceptor, the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA. SrSHT exhibited the same substrate specificity as SmSHT, yet exhibited significantly higher catalytic activity than SmSHT. For example, under caffeoyl-CoA and spermidine, Kcat of SrSHT was 37.3% higher than SmSHT. Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures. In particular, the conserved Lys402 adjacent to the DFGWG motif, and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT. These substitutions may contribute to the enhanced activity in SrSHT. Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.

A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants

“…qRT-PCR was performed in triplicates using GAPDH as a constitutive control. Note that SHT1 corresponded to SHT previously reported [26], while SHT4 was renamed after SpmHT later. Relative expression was shown in fold changes (lowest value = 1).…”

“…2). Note that SHT1 is reported by us previously [26]. However, SHT4 was detected predominantly in fruit from S. richardii , and hardly detected in eggplant fruit, strongly suggesting that it is a SpmHT candidate.…”

“…Nicotiana attenuata SHT (NaDH29) and putrescine HT (NaAT1) were able to catalyze the synthesis of HCSpd and HCPut, respectively [25]. Previously we identified an eggplant SHT (SmSHT) that was predicted to play a major role in the formation of HCSpd in flowers and fruit [26]. However, an HT specifically responsible for the biosynthesis of HCSpm has yet to be identified.

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Candidate gene mapping identifies genomic variations in the fire blight susceptibility genes HIPM and DIPM across the Malus germplasm

“…The lack of phenotypic information for accessions in this dataset hindered the possibility of making an association analysis between SNP markers and fire blight susceptibility. Thus, amplicon sequencing was employed to amplify and assess the genetic diversity in the HIPM and DIPM genes in 93 selected apple accessions (collection 2; Supplementary Table S1 ) 44 . The latter analysis identified a total of 87 SNP variants across 9 DIPM and HIPM genes ranging from 4 ( HIPM1b ) to 16 ( DIPM2a ) (Table 1 ).…”

“…Total genomic DNA from Malus accessions of Collection 2 (Supplementary Table S1 ) was extracted from young fresh leaves using the Wizard Genomic DNA Purification Kit (Promega, USA), according to the manufacturer's instructions. The concentration of extracted DNA was analyzed with a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Gand Island, NY, USA) and the quality of DNA samples was assessed by running a 0.8% agarose gel at 80 V for 1.5 h. Genomic DNA was amplified with the previously designed and selected primer pairs (Supplementary Table S2 ) to amplify amplicon pools, which were sequenced on the Illumina MiSeq Platform (Illumina, San Diego, CA, USA), according to the AmpSeq protocol at the Genomics Platform Facility of Cornell University (Ithaca, NY, USA) 44 .…”