L.pone.0048006.gmature seeds. During early stages of seedling development sinapine is converted to sinapoylmalate via sinapate and sinapoylglucose [46,47]. Sinapoylmalate protects plant leaves from 
UV-B irradiation [12,48?1] and is involved in UV-Binduced defense against fungi in A. thaliana leaves [52]. On the other hand, much experimental evidence suggests that the sinapine stored in rapeseed provides a supply of sinapate and choline, both of which serve as important precursors for essential plant components. Sinapine (12) degrades into sinapate and choline during early stages of seed germination [6,53,54], and the two components are used in later biosynthetic processes [53]. In Raphanus sativus seedlings, choline released from sinapine was proven to be processed biosynthetically to phosphatidylcholine [6], and the sinapic acid moiety was hypothesized as the precursor for the biosynthesis of further phenolic compounds, such as flavonoids [53]. Thus, all 4-IBP web products released or converted from sinapine during early steps of seed germination (sinapoylglucose, sinapoylmalate, sinapate and choline) play essential physiological and ecological roles for the seedling and plant [5]. The even distribution of sinapine in rapeseed embryo tissue supports its depot function.Figure 4. Distribution of the major MedChemExpress BTZ-043 cyclic spermidine in rapeseed. (A) Structure of the major cyclic spermidine conjugate (13) identified from rapeseed. (B) The concentration of 13 in different tissues and whole rapeseed. HR, hypocotyl and radicle; IC, inner cotyledon; OC, outer cotyledon; and SE, seed coat and endosperm. Each column shows the mean of four replicates with standard error, and *means not detectable. doi:10.1371/journal.pone.0048006.gCyclic Spermidine Conjugates in RapeseedCyclic spermidine conjugates in non-glucosinolate (NG) fractions of laser-microdissected rapeseed tissues were detected by HPLC-ESIMS in positive ionization mode (see Materials and methods). The major peak in extracted ion chromatogram (EIC) for ions at m/z 496.4 ([M+H]+) (Figure S1) was identified as 
the major cyclic spermidine conjugate (13) (Figure 4A), based on its molecular mass of 495 Da and comparing the retention time with the compound recently isolated from rapeseed (unpublished data). Based on the same molecular mass in the EIC and the same fragmentation patterns in MS/MS analysis compared to those of the major peak, several minor peaks (Figure S1) were suggested to be isomeric cyclic spermidine conjugates. However, structural details remained unassigned because nuclear magnetic resonance (NMR) data are lacking. The average concentration of compound 13 in the whole rapeseed is 1.94 mmol/g, as calculated from a calibration curve. Interestingly, the cyclic spermidine conjugates were found only in HR, where the average concentration of 13 isas high as 13.48 mmol/g. Compound 13 and minor cyclic spermidines are absent in SE, IC and OC tissues (Figures 4B, S1). No free spermidine was detected in any sample. Polyamines (PAs) and phenylpropanoid-polyamine conjugates (PPCs) are widely distributed 16574785 in plants [55], including seeds [56], and play important roles in plant growth, abiotic stress tolerance and defense against insect herbivores [57?9]. Compound 13 (Figure 4A) was previously identified as the sole PPC from the same plant material, rapeseed [47,60]. Nevertheless, this is the first time that the distribution of PPCs in seeds has been directly demonstrated. Our results showed that PPCs in rape.L.pone.0048006.gmature seeds. During early stages of seedling development sinapine is converted to sinapoylmalate via sinapate and sinapoylglucose [46,47]. Sinapoylmalate protects plant leaves from UV-B irradiation [12,48?1] and is involved in UV-Binduced defense against fungi in A. thaliana leaves [52]. On the other hand, much experimental evidence suggests that the sinapine stored in rapeseed provides a supply of sinapate and choline, both of which serve as important precursors for essential plant components. Sinapine (12) degrades into sinapate and choline during early stages of seed germination [6,53,54], and the two components are used in later biosynthetic processes [53]. In Raphanus sativus seedlings, choline released from sinapine was proven to be processed biosynthetically to phosphatidylcholine [6], and the sinapic acid moiety was hypothesized as the precursor for the biosynthesis of further phenolic compounds, such as flavonoids [53]. Thus, all products released or converted from sinapine during early steps of seed germination (sinapoylglucose, sinapoylmalate, sinapate and choline) play essential physiological and ecological roles for the seedling and plant [5]. The even distribution of sinapine in rapeseed embryo tissue supports its depot function.Figure 4. Distribution of the major cyclic spermidine in rapeseed. (A) Structure of the major cyclic spermidine conjugate (13) identified from rapeseed. (B) The concentration of 13 in different tissues and whole rapeseed. HR, hypocotyl and radicle; IC, inner cotyledon; OC, outer cotyledon; and SE, seed coat and endosperm. Each column shows the mean of four replicates with standard error, and *means not detectable. doi:10.1371/journal.pone.0048006.gCyclic Spermidine Conjugates in RapeseedCyclic spermidine conjugates in non-glucosinolate (NG) fractions of laser-microdissected rapeseed tissues were detected by HPLC-ESIMS in positive ionization mode (see Materials and methods). The major peak in extracted ion chromatogram (EIC) for ions at m/z 496.4 ([M+H]+) (Figure S1) was identified as the major cyclic spermidine conjugate (13) (Figure 4A), based on its molecular mass of 495 Da and comparing the retention time with the compound recently isolated from rapeseed (unpublished data). Based on the same molecular mass in the EIC and the same fragmentation patterns in MS/MS analysis compared to those of the major peak, several minor peaks (Figure S1) were suggested to be isomeric cyclic spermidine conjugates. However, structural details remained unassigned because nuclear magnetic resonance (NMR) data are lacking. The average concentration of compound 13 in the whole rapeseed is 1.94 mmol/g, as calculated from a calibration curve. Interestingly, the cyclic spermidine conjugates were found only in HR, where the average concentration of 13 isas high as 13.48 mmol/g. Compound 13 and minor cyclic spermidines are absent in SE, IC and OC tissues (Figures 4B, S1). No free spermidine was detected in any sample. Polyamines (PAs) and phenylpropanoid-polyamine conjugates (PPCs) are widely distributed 16574785 in plants [55], including seeds [56], and play important roles in plant growth, abiotic stress tolerance and defense against insect herbivores [57?9]. Compound 13 (Figure 4A) was previously identified as the sole PPC from the same plant material, rapeseed [47,60]. Nevertheless, this is the first time that the distribution of PPCs in seeds has been directly demonstrated. Our results showed that PPCs in rape.
