Salicornia ramosissima (Hook. f.) W. A. Clarke & E. S. Marshall

Maciel, Elisabete, Lillebø, Ana, Domingues, Pedro, Costa, Elisabete da, Calado, Ricardo & Domingues, M. Rosário M., 2018, Polar lipidome profiling of Salicornia ramosissima and Halimione portulacoides and the relevance of lipidomics for the valorization of halophytes, Phytochemistry 153, pp. 94-101 : 95-96

2.1. Lipid profile of Salicornia ramosissima

Seventeen different fatty acids (FAs) were identified in the lipid extracts of S. ramosissima ( Table 1 ). Results obtained indicate that S. ramosissima had a high content of long chain FA, with a length between 16 and 18 carbons and a high level of unsaturation (e.g., polyunsaturated fatty acids (PUFA)) up to 61.3%. PUFA included mainly n -3 and n -6 FA. The most abundant FAs were α -linolenic acid (C18:3 n -3, ALA), which correspond to with nearly 40% of the total pool of FAs, followed by linoleic acid (C18:2 n -6, LA), which contributed with 20% of the total pool. Saturated fatty acids (SFA) were the second most abundant group of FAs, whose content was estimated to be ∼30% of total FAs present in S. ramosissima . The main saturated FA was palmitic acid (C16:0, PA) (22.1% of the total pool of FAs). Monounsaturated fatty acids (MUFA) were less abundant than PUFA and SFA, only accounting for approximately 6% of the total pool of FA. Oleic acid (C18:1 n -9) was the most abundant MUFA, although it only accounted for 5% of the total pool of FAs.

The polar lipidome was determined in crude lipid extracts using HILIC coupled to a high resolution and accurate mass spectrometer. Over 200 molecular species were identified in S. ramosissima lipid extracts, distributed by five different classes of PL, three classes of glycolipids and one glycosphingolipid, as described above. The MS profiles are shown in supplementary information ( Figs. S1 and S2).

MS/MS data allowed the identification of 62 PC, 43 PE, 30 PA, 15 PG and 9 PI molecular species, with different fatty acyl moieties ranging from 15 to 24 carbon atoms and containing from 0 to 6 double bonds. FA distribution was found to be class dependent ( Table 2). The main PC molecular species were found at m/z 756.551 (PC 16:0/18:3), m/z 758.567 (PC 16:0/18:2), m/z 780.550 (PC 18:2/18:3), m/z 782.566 (PC 18:2/18:2 and PC 18:1/18:3) and m/z 870.666 (PC 18:2/ 24:0). The PC profile of S. ramosissima was characterized by the presence of polyunsaturated PC species, odd-chain FA and very long FA, such as C24:0. These FA were also detected using GC-MS analysis ( Table 1 ). In the case of PE class, the molecular species were composed of a combination of acyl groups ranging from 15 to 24 carbon atoms containing 0 to 3 double bonds ( Table 2). The most abundant PE molecular species was found at m/z 714.490, corresponding to PE 16:0/ 18:2, followed by PE 18:2/18:2 at m/z 738.505. Similar to the PC class, some species with very long chain FAs, such as 24:0 were identified in PE class, namely at m/z 824.614, m/z 826.630, m/z 858.584 and m/z 872.600. PE molecular species esterified with odd-chain FA were also found such as the FA C15:0, C19:2 and C21:0. PA class included molecular species carrying fatty acyl groups ranging from 16 to 19 carbon atoms and the major PA molecular species were identified at m/z 669.448 (PA 16:0/18:3 and PA 16:1/18:2), m/z 671.464 (PA 16:0/ 18:2), m/z 693.448 (PA 18:2/18:3) and m/z 695.463 (PA 18:2/18:2 and PA 18:1/18:3. Species with odd-chain fatty acids (C19:0, C19:1, C19:2, C19:3, C17:0 and C21:0) were also identified. In the case of PG class, the most abundant molecular species were found at m/z 719.484 and m/z 745.499 ( Fig. S 1 in the supplementary information) corresponding to PG 16:0/16:1 and PG 16:0/18:2, respectively. The acyl composition of PG ranged from 16 to 20 carbon atoms. The molecular species identified in the case of PI class included only the fatty acyl chains of C16:0, C18:0, C18:1, C18:2 and C18:3. The most abundant PI molecular specie was found at m/z 833.515 and was assigned to PI 16:0/18:2.

Table 2 Phospholipid molecular species identified by HILIC− Q− Exactive− MS from total lipid extract of Salicornia ramosissima .

The analysis and interpretation of LC− MS/MS data allowed to identify 13 molecular species of SQDG, 21 MGDG, 10 DGDG and 5 HexCer. The acyl composition of GL ranged from 16 to 22 carbon atoms and 0 to 3 double bonds ( Table 3 ). The main SQDG molecular species was SQDG 18:3/16:0 at m/z 815.496, followed by SQDG 16:0/ 16:0 at m/z 793.511 and SQDG 18:3/18:3 at m/z 837.479. For MGDG, the major molecular specie was MGDG 18:3/18:3 at m/z 792.56. Some MGDG molecular species with odd-chain FA were observed, namely with FA C17:0, C19:1, C19:2, C19:3 and C21:0. The fatty acyl chains of DGDG ranged from 16 to 21 carbon atoms. The main molecular specie of DGDG class was the DGDG 18:3/18:3 at m/z 954.612. Two DGDG molecular species were identified with odd-chain FA: DGDG 19:2/18:3 and DGDG 21:0/18:3 at m/z 970.607 and 1002.596 respectively.

The glycosphingolipids HexCer were also identified and included molecular species with both di- and trihydroxy long-chain bases. The sphingoid bases identified in the present work were C18, while the length of the identified hydroxylated fatty acids varied between 16 and 24 carbon atoms. The most abundant molecular species was HexCer t 18:1/h24:0 detected at m/z 844.684 with a trihydroxy long chain base. The HexCer d 18:1/16:1, at m/z 714.549, displayed a dihydroxy long chain base moiety.

Figures

Fig. 1. Potential pathways for the valorization of Salicornia ramosissima and Halimione portulacoides fostered by high resolution and high throughput analysis of their lipidome. Top pie chart refers to the percentage of saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (FA) present in the edible parts of the two halophytes species; n-3 and n-6 percentage refer solely to PUFA. Phospholipids (PL) and glycolipids (GL) determined through liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS) are represented as the number of molecular species identified on each class, namely phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), hexosyl ceramide (HexCer) sulfoquinovosyldiacylglycerol (SQDG), digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (DGDG).

Tables

Table 2 Phospholipid molecular species identified by HILIC−Q−Exactive−MS from total lipid extract of Salicornia ramosissima. [M + H]+ Phosphatidylcholine 709.443 PA 18:3/19:1; 18:2/19:2 732.550 PC 16:0/16:1 711.459 PA 19:1/18:2 734.542 PC 16:0/16:0 723.421 PA 20:1/18:3; 20:2/18:2 756.551 PC 16:0/18:3 725.438 PA 20:0/18:3; 20:1/18:2 758.567 PC 16:0/18:2 727.453 PA 20:0/18:2 760.579 PC 34:1 739.419 PA 21:0/18:3 762.588 PC 16:0/18:0 741.433 PA 21:0/18:2 778.534 PC 18:3/18:3 [M − H]− Phosphatidylethanolamine 780.550 PC 18:2/18:3 700.490 PE 15:0/18:2; 16:0/17:2 782.566 PC 18:2/18:2; 18:1/18:3 712.490 PE 16:0/18:3 784.579 PC 36:3 714.490 PE 16:0/18:2 786.597 PC 18:0/18:2; 18:1/18:1 730.500 PE 16:0/19:1; 17:0/18:1 798.536 PC 18:3/19:0; 18:2/19:1; 18:1/19:2 734.474 PE 18:3/18:3 788.608 PC 36:1 736.489 PE 18:2/18:3 796.547 PC 18:4/19:0; 18:3/19:1; 18:2/19:2 738.505 PE 18:2/18:2 800.612 PC 37:2 740.517 PE 18:0/18:3; 18:1/18:2 802.532 PC 18:4/20:4 742.537 PE 18:0/18:2; 18:1/18:1; 17:0/19:2 744.450 PE 18:0/18:1; 16:0/20:1 804.548 PC 38:7 746.495 PE 16:0/20:0 806.562 PC 18:3/20:3 752.484 PE 18:2/19:2 772.581 PC 16:0/19:2; 17:0/18:2 750.504 PE 18:3/19:2; 18:2/19:3 808.580 PC 18:3/20:2; 18:2/20:3 754.499 PE 18:2/19:1 810.597 PC 18:3/20:1; 18:2/20:2 760.474 PE 16:0/21:0 812.613 PC 18:3/20:0; 18:2/20:1; 18:1/20:2 766.536 PE 18:3/20:1; 18:2/20:2 814.630 PC 18:2/20:0; 18:1/20:1; 19:1/19:1; 19:0/19:2 768.551 PE 18:2/20:1; 18:3/20:0; 18:1/20:2 816.618 PC 18:1/20:0 770.494 PE 18:2/20:0 826.628 PC 21:0/18:3; 18:2/21:1 782.493 PE 18:3/21:0; 19:2/20:1 828.645 PC 21:0/18:2 784.474 PE 18:2/21:0; 19:2/20:0 836.610 PC 20:2/20:3 798.598 PE 22:0/18:2 838.603 PC 22:2/18:2; 22:1/18:3 810.493 PE 23:0/18:3; 18:2/23:1 840.618 PC 22:0/18:3; 22:1/18:2 812.614 PE 23:0/18:2 842.629 PC 22:0/18:2 816.536 PE 16:0/25:0; 20:0/21:0 856.654 PC 23:0/18:2 824.614 PE 18:3/24:0 868.651 PC 24:0/18:3 826.630 PE 18:2/24:0 870.666 PC 24:0/18:2 844.568 PE 21:0/22:0 872.678 PC 24:0/18:1 858.584 PE 20:0/24:0 886.694 PC 44:8 872.600 PE 21:0/24:0 884.683 PC 25:0/18:2 [M − H]− Phosphatidylglycerol 890.634 PC 44:6 719.484 PG 16:0/16:1 892.650 PC 44:5 741.468 PG 16:1/18:3; 16:0/18:4 894.665 PC 44:4 743.484 PG 34:3 898.697 PC 44:2 745.499 PG 16:0/18:2 924.714 PC 46:3 757.462 PG 16:1/19:2; 16:0/19:3; 17:0/18:3 926.728 PC 46:2 765.471 PG 18:3/18:3 [M − H]− Phosphatidic acid 767.483 PG 18:2/18:3 645.448 PA 16:0/16:1 773.53 PG 18:0/18:2; 16:1/20:1; 16:0/20:2; 18:1/18:1 667.433 PA 16:1/18:3 777.55 PG 16:0/20:0 669.448 PA 16:0/18:3; 16:1/18:2 [M − H]− Phosphatidylinositol 671.464 PA 16:0/18:2 831.499 PI 16:0/18:3 683.427 PA 17:0/18:3 833.515 PI 16:0/18:2 685.443 PA 16:0/19:2; 17:0/18:2 853.481 PI 18:3/18:3 687.458 PA 16:0/19:1; 16:1/19:0 855.499 PI 18:2/18:3 691.432 PA 18:3/18:3 859.53 PI 18:0/18:3; 18:1/18:2 693.448 PA 18:2/18:3 861.546 PI 18:0/18:2; 18:1/18:1 695.463 PA 18:2/18:2; 18:1/18:3 863.552 PI 18:0/18:1 699.495 PA 18:0/18:2; 18:1/18:1 701.438 PA 16:0/20:1 703.452 PA 16:0/20:0 707.427 PA 18:3/19:2; 18:2/19:3

[M + H] +	Phosphatidylcholine	709.443	PA 18:3/19:1; 18:2/19:2
732.550	PC 16:0/16:1	711.459	PA 19:1/18:2
734.542	PC 16:0/16:0	723.421	PA 20:1/18:3; 20:2/18:2
756.551	PC 16:0/18:3	725.438	PA 20:0/18:3; 20:1/18:2
758.567	PC 16:0/18:2	727.453	PA 20:0/18:2
760.579	PC 34:1	739.419	PA 21:0/18:3
762.588	PC 16:0/18:0	741.433	PA 21:0/18:2
778.534	PC 18:3/18:3	[M − H] −	Phosphatidylethanolamine
780.550	PC 18:2/18:3	700.490	PE 15:0/18:2; 16:0/17:2
782.566	PC 18:2/18:2; 18:1/18:3	712.490	PE 16:0/18:3
784.579	PC 36:3	714.490	PE 16:0/18:2
786.597	PC 18:0/18:2; 18:1/18:1	730.500	PE 16:0/19:1; 17:0/18:1
798.536	PC 18:3/19:0; 18:2/19:1; 18:1/19:2	734.474	PE 18:3/18:3
788.608	PC 36:1	736.489	PE 18:2/18:3
796.547	PC 18:4/19:0; 18:3/19:1; 18:2/19:2	738.505	PE 18:2/18:2
800.612	PC 37:2	740.517	PE 18:0/18:3; 18:1/18:2
802.532	PC 18:4/20:4	742.537	PE 18:0/18:2; 18:1/18:1; 17:0/19:2
		744.450	PE 18:0/18:1; 16:0/20:1
804.548	PC 38:7	746.495	PE 16:0/20:0
806.562	PC 18:3/20:3	752.484	PE 18:2/19:2
772.581	PC 16:0/19:2; 17:0/18:2	750.504	PE 18:3/19:2; 18:2/19:3
808.580	PC 18:3/20:2; 18:2/20:3	754.499	PE 18:2/19:1
810.597	PC 18:3/20:1; 18:2/20:2	760.474	PE 16:0/21:0
812.613	PC 18:3/20:0; 18:2/20:1; 18:1/20:2	766.536	PE 18:3/20:1; 18:2/20:2
814.630	PC 18:2/20:0; 18:1/20:1; 19:1/19:1; 19:0/19:2	768.551	PE 18:2/20:1; 18:3/20:0; 18:1/20:2
816.618	PC 18:1/20:0	770.494	PE 18:2/20:0
826.628	PC 21:0/18:3; 18:2/21:1	782.493	PE 18:3/21:0; 19:2/20:1
828.645	PC 21:0/18:2	784.474	PE 18:2/21:0; 19:2/20:0
836.610	PC 20:2/20:3	798.598	PE 22:0/18:2
838.603	PC 22:2/18:2; 22:1/18:3	810.493	PE 23:0/18:3; 18:2/23:1
840.618	PC 22:0/18:3; 22:1/18:2	812.614	PE 23:0/18:2
842.629	PC 22:0/18:2	816.536	PE 16:0/25:0; 20:0/21:0
856.654	PC 23:0/18:2	824.614	PE 18:3/24:0
868.651	PC 24:0/18:3	826.630	PE 18:2/24:0
870.666	PC 24:0/18:2	844.568	PE 21:0/22:0
872.678	PC 24:0/18:1	858.584	PE 20:0/24:0
886.694	PC 44:8	872.600	PE 21:0/24:0
884.683	PC 25:0/18:2	[M − H] −	Phosphatidylglycerol
890.634	PC 44:6	719.484	PG 16:0/16:1
892.650	PC 44:5	741.468	PG 16:1/18:3; 16:0/18:4
894.665	PC 44:4	743.484	PG 34:3
898.697	PC 44:2	745.499	PG 16:0/18:2
924.714	PC 46:3	757.462	PG 16:1/19:2; 16:0/19:3; 17:0/18:3
926.728	PC 46:2	765.471	PG 18:3/18:3
[M − H] −	Phosphatidic acid	767.483	PG 18:2/18:3
645.448	PA 16:0/16:1	773.53	PG 18:0/18:2; 16:1/20:1; 16:0/20:2; 18:1/18:1
667.433	PA 16:1/18:3	777.55	PG 16:0/20:0
669.448	PA 16:0/18:3; 16:1/18:2	[M − H] −	Phosphatidylinositol
671.464	PA 16:0/18:2	831.499	PI 16:0/18:3
683.427	PA 17:0/18:3	833.515	PI 16:0/18:2
685.443	PA 16:0/19:2; 17:0/18:2	853.481	PI 18:3/18:3
687.458	PA 16:0/19:1; 16:1/19:0	855.499	PI 18:2/18:3
691.432	PA 18:3/18:3	859.53	PI 18:0/18:3; 18:1/18:2
693.448	PA 18:2/18:3	861.546	PI 18:0/18:2; 18:1/18:1
695.463	PA 18:2/18:2; 18:1/18:3	863.552	PI 18:0/18:1
699.495	PA 18:0/18:2; 18:1/18:1
701.438	PA 16:0/20:1
703.452	PA 16:0/20:0
707.427	PA 18:3/19:2; 18:2/19:3