Arabidopsis Leaf Chloroplasts Have a Specific Sphingolipidome

Sphingolipids are ubiquitous in eukaryotes and certain prokaryotes, where they serve as vital components of biological membranes and bioactive molecules. Chloroplasts have complex membrane structures that play crucial roles in photosynthesis, but their specific sphingolipidome remains unreported. In this study, we used liquid chromatography–mass spectrometry (LC-MS/MS) to analyze the sphingolipidome of purified Arabidopsis thaliana chloroplasts. We detected 92 chloroplast sphingolipids. The chloroplast sphingolipidome differed from total leaf (TL) samples, with a higher content of free long-chain bases and hydroxyceramides and a greater proportion of complex sphingolipids with 16C fatty acid (FA) forms. Notably, chloroplast glucosylceramides were predominantly the d18:1 h16:0 and t18:1 h16:0 forms rather than the 24C FA form found in TL and other cellular structures. Comparing the sphingolipidomes of different cellular structures underscores the inhomogeneity of the intracellular distribution of sphingolipids. This provides a robust reference for further elucidating the function of sphingolipids in plant cells.


Introduction
Sphingolipids are ubiquitous in eukaryotes and certain prokaryotes that are composed of long-chain base (LCB) and fatty acid (FA) chains [1,2].They are essential constituents of biological membranes, where they maintain the membrane's structural integrity and biophysical properties [3,4].Moreover, they serve as bioactive molecules, playing pivotal roles in plant growth, development, and responses to biotic and abiotic stresses.These functions include the regulation of cell death, organ development, resistance to abiotic stresses (salt, drought, and cold), defense against pathogens, and the promotion of rhizobial symbiosis [5][6][7][8][9][10].
The structural diversity of sphingolipids results from the variation in LCB species, FA chain lengths, hydroxylation, unsaturation, and modifications of the head group [2].The LCB can be conjugated with FA chains and polar head groups to form complex sphingolipids.The common complex sphingolipids in plant cells include ceramide (Cer), hydroxyceramide (hCer), glucosylceramide (GlcCer), and glycosylinositolphosphoceramide (GIPC), with GIPC being the most abundant sphingolipid in plant cells [11].
The majority of enzymes involved in sphingolipid biosynthesis and metabolism localize in the endoplasmic reticulum (ER) and Golgi apparatus, and their products are subsequently transferred and transported to other cellular locations [12,13].The advent of liquid chromatography-mass spectrometry (LC-MS/MS) technology has facilitated the high-resolution identification of plant sphingolipids [14].Studies have provided detailed descriptions of the sphingolipidome at the level of the plant strain and organ, as well as its dynamic changes under varying conditions [15,16].
Sphingolipids are not uniformly distributed in cellular membrane systems [13].However, our understanding of the sphingolipidome in various membrane-containing organelles of plants remains incomplete.Previous studies have reported that GlcCer and Plants 2024, 13, 299 2 of 15 GIPC constitute 40% of the plasma membrane (PM) lipids [17,18], and sphingolipids account for approximately 20% of the lipids in ER and Golgi membranes, playing a crucial role in maintaining their morphology and function [19][20][21][22].A recent study determined that the sphingolipid composition of vacuolar membranes (VM) differs from that of the PM, detergent-resistant membrane (DRM), and microsomal membrane (MIC), with a higher proportion of GlcCer rather than GIPC [23].Additionally, the sphingolipid composition of plant mitochondria (Mito) has recently been reported to differ from that of other organelles and membrane systems [24].These findings suggest that plant organelles have distinct sphingolipidomes.
Chloroplasts and their thylakoids constitute the most expansive membrane system in leaf mesophyll cells and are abundant in lipids [25].Additionally, a rigorously regulated mechanism traffics lipids between the outer membrane of the chloroplast and the ER, underscoring the complexity and significance of lipid dynamics within plant cells [26].However, the sphingolipidome of chloroplasts has yet to be reported.In this study, we delineate the sphingolipidome of chloroplasts in Arabidopsis using LC-MS/MS.We illuminate the unique characteristics of the sphingolipid composition within the chloroplast and show that it can be distinguished from that of the total leaf or other organelles.

Purification of Chloroplast Fractions
In this study, we characterized the sphingolipid composition of the total leaf (TL) and chloroplast samples.The sphingolipidome of the total leaf has been widely reported [14].We, therefore, used total leaf sphingolipid data as controls to compare against chloroplasts.The procedure for chloroplast isolation, which has been simplified for the purposes of this experiment, is depicted in Figure 1a.To assess the purity of the isolated chloroplast fraction, we employed immunoblotting assays using several marker proteins.These were as follows: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) for the plasma membrane, luminal BINDING PROTEIN 2 (BIP2) for the endoplasmic reticulum (ER), the Rubisco large subunit (RbcL) for the chloroplast, H(+)-ATPASE for the vacuole (V-ATPase), VOLTAGE-DEPENDENT ANION CHANNEL 1 (VDAC1) for the mitochondrion, and histone H3 for the nucleus.All these marker proteins were detectable in the TL samples.However, in the chloroplast samples, only RbcL was detected, and it was found to be enriched (Figure 1b).These findings suggest that the chloroplast samples used in this study were of high purity.

Distribution of Sphingolipid Classes
The numbers of five sphingolipid classes (LCBs, Cers, hCers, GlcCers, and GIPCs) were expressed in terms of the absolute content (per mg protein of chloroplasts and per g dry weight (DW) of leaves) (Supplementary Table S1) and relative content (molecular percentage, mol %) (Supplementary Table S2).In terms of absolute sphingolipid content, LCBs, Cers, hCers, and GlcCers in chloroplasts were present in similar amounts, ranging from 1.5 to 10 nmol/mg protein, while GIPCs were present in significantly higher amounts of approximately 30 nmol/mg protein (Figure 3a).
We used relative content (mol %) for all the next comparisons to ensure that the data of chloroplast and TL sphingolipids were the same condition (Supplementary Table S2).This revealed notable differences between the chloroplast and TL samples (Figure 3b).GIPCs were overwhelmingly predominant in both chloroplast and TL samples, constituting about 60% to 80% of the total sphingolipid content.GlcCers were equally present in all samples, from 10% to 14%.In TL samples, LCBs constituted only about 1% of the total content, while in chloroplast samples, they accounted for a larger proportion of about 10%.Similarly, the proportion of Cers and hCers in TL samples was low (3% to 5%), while in chloroplast samples, the proportion of hCers was about 11%.These findings suggest a different distribution of sphingolipid classes between the chloroplasts and total leaf samples.
Plants 2024, 13, x FOR PEER REVIEW Figure 1.Experimental procedure for the sphingolipid analysis and purity ass experimental approach to obtain total leaf (TL) and chloroplast samples from Arabi sphingolipid analysis.Yellow asterisks indicate a target precipitation containing Immunodetection of BAK1 from the plasma membrane, BIP2 from the endoplasmic from chloroplasts, V-ATPase from vacuoles, VDAC1 from the mitochondria, and nuclei.Proteins were separated using SDS-PAGE and detected via immunobl biological replicates were performed for immunodetection with all protein markers as a loading control.

Overall Assessment of Sphingolipid Profiles
The sphingolipid profiles, encompassing LCBs and complex sphin hCers, GlcCers, and GIPCs) (Figure 2a), were comprehensively analyz MS/MS.To ensure the reliability of the results, components exhibiting shapes or low-peak values were deemed to be undetected.We identifi  (c) Heatmap plots of the abundance of LCBs, Cers, hCers, GlcCers, and GIPCs.The Z-score represents the standardized content of individual sphingolipid species in mol %.The scale from the left value to the right value represents the number of standard deviations from the average of each row (i.e., sample types), with red indicating higher contents than average and the blue color indicating lower contents than average.An "O" in the heatmap means that sphingolipid species were not detected in this sample group.The sphingolipid species that were not detected in any of the samples are not shown.

Distribution of Sphingolipid Classes
The numbers of five sphingolipid classes (LCBs, Cers, hCers, GlcCers, and GIPCs) were expressed in terms of the absolute content (per mg protein of chloroplasts and per g Multivariate analysis and heatmap plots of sphingolipid profiles.(a) Representation of the structure of main complex sphingolipid classes.The R1 in the structure of GIPC indicates mannose, glucosamine, N-acetyglucosamine, galactose, arabinose, or a combination of these hexose groups as described in [27].(b) Partial least squares-discriminant analysis (PLS-DA) of sphingolipid profiles of the chloroplast and total leaf samples.The analysis is based on the relative content (mol %) of all detected sphingolipid species.In this study, nine individual plants were set for each sample type.(c) Heatmap plots of the abundance of LCBs, Cers, hCers, GlcCers, and GIPCs.The Z-score represents the standardized content of individual sphingolipid species in mol %.The scale from the left value to the right value represents the number of standard deviations from the average of each row (i.e., sample types), with red indicating higher contents than average and the blue color indicating lower contents than average.An "O" in the heatmap means that sphingolipid species were not detected in this sample group.The sphingolipid species that were not detected in any of the samples are not shown.
of the total content, while in chloroplast samples, they accounted for a larger p of about 10%.Similarly, the proportion of Cers and hCers in TL samples was l 5%), while in chloroplast samples, the proportion of hCers was about 11%.Thes suggest a different distribution of sphingolipid classes between the chloroplast leaf samples.
To assess the specific composition of sphingolipids in complex sphingolipids (Cers, hCers, GlcCers, GIPCs), we considered four LCBs (t18:0, t18:1, d18:0, d18:1) and ten FAs with lengths ranging from 16 to 26C (16:0, 18:0, 20:0, 20:1, 22:0, 22:1, 24:0, 24:1, 26:0, 26:1) which differ in length, degree of unsaturation, and C2-hydroxylation.For the LCB composition of complex sphingolipids, the predominant form was trihydroxylated (t18) and unsaturated forms in both the chloroplast and TL samples (Figure 4a,b).However, when considering this proportion, chloroplast samples differed substantially from the TL samples.Specifically, the proportion of t18 forms was higher in chloroplast samples than in TL for Cer, hCer, and GIPC classes but lower for the GlcCer class (Figure 4b).Concurrently, unsaturated forms were higher in chloroplast samples than in TL, especially in the GlcCer class (Figure 4c).Concurrently, unsaturated forms were higher in chloroplast samples than in TL, especially in the GlcCer class (Figure 4c).In terms of the FA composition, all classes, with the exception of GlcCer in chloroplast samples, were predominantly composed of very long-chain fatty acids (VLCFAs) (20C, 22C, 24C, 26C) as opposed to long-chain fatty acids (LCFAs) (16C and 18C) (Figure 5b).Notably, Cer, hCer, and GlcCer in chloroplast samples were composed of a higher proportion of the 16C form than in TL, particularly GlcCer, which contained more than 50% of the 16C form and was the only sphingolipid class with an LCFA/VLCFA ratio greater than one (Figure 5b).Conversely, GIPC in chloroplasts contained a lower proportion of the 16C form, accompanied by a higher proportion of 24C and 26C forms than TL (Figure 5a,b).Calculating the ratio of unsaturated to saturated FA showed that all sphingolipid classes were dominated by unsaturated FA forms, but the proportion of saturated forms in GlcCer was significantly higher in chloroplast samples compared to TL (Figure 5c).Collectively, LCB and FA compositions exhibited notable differences between In terms of the FA composition, all classes, with the exception of GlcCer in chloroplast samples, were predominantly composed of very long-chain fatty acids (VLCFAs) (20C, 22C, 24C, 26C) as opposed to long-chain fatty acids (LCFAs) (16C and 18C) (Figure 5b).Notably, Cer, hCer, and GlcCer in chloroplast samples were composed of a higher proportion of the 16C form than in TL, particularly GlcCer, which contained more than 50% of the 16C form and was the only sphingolipid class with an LCFA/VLCFA ratio greater than one (Figure 5b).Conversely, GIPC in chloroplasts contained a lower proportion of the 16C form, accompanied by a higher proportion of 24C and 26C forms than TL (Figure 5a,b).Calculating the ratio of unsaturated to saturated FA showed that all sphingolipid classes were dominated by unsaturated FA forms, but the proportion of saturated forms in GlcCer was significantly higher in chloroplast samples compared to TL (Figure 5c).Collectively, LCB and FA compositions exhibited notable differences between the chloroplast and TL samples, indicating that chloroplasts have a unique sphingolipid composition.

Predominant Sphingolipid Species
We detected a considerable number of sphingolipid species, but many of them were present in low abundance (Supplementary Table S2).To further scrutinize the potentially significant sphingolipid species, we classified those with a relative content exceeding 1% as abundant species.This classified a total of 49 species, including 16 species of Cers, 14 species of hCers, 9 species of GlcCers, and 14 species of GIPCs as abundant species (Figure 6).

Predominant Sphingolipid Species
We detected a considerable number of sphingolipid species, but many of them were present in low abundance (Supplementary Table S2).To further scrutinize the potentially significant sphingolipid species, we classified those with a relative content exceeding 1% as abundant species.This classified a total of 49 species, including 16 species of Cers, 14 species of hCers, 9 species of GlcCers, and 14 species of GIPCs as abundant species (Figure 6).
Although the predominant species of the above three sphingolipid classes were consistent in chloroplasts and TL, the most abundant species of GlcCer showed a clear difference between the chloroplast and TL samples.In TL, the t18:1 h24:1 form was the predominant species, while in chloroplast samples, t18:0 h16:0 and d18:1 h16:0 forms were the predominant species (Figure 6c).This further underscores the specificity of chloroplast sphingolipid composition.

Discussion
This study presents the detailed identification of the sphingolipid composition of Arabidopsis chloroplasts.The sphingolipidome of the total leaves (TL) identified in our study aligns with those reported in previous studies [14,28].When compared to other Among the abundant Cer species, t18:1 c24:0 and t18:1 c26:0 were the most abundant in both chloroplast and TL samples, accounting for 40%~50% of the total (Figure 6a).For the hCer class, both sample types were overwhelmingly dominated by t18:1 h24:0 and t18:1 h24:1, constituting approximately 50% of the total, and a greater proportion of t18:1 h16:0 and d18:1 h16:0 was involved in the chloroplast sphingolipid assembly (Figure 6b).GIPC was also dominated by forms of t18:1 h24:0 and t18:1 h24:1, with the proportion of the t18:1 h24:0 form in chloroplasts being about 10% higher than that in TL (Figure 6d).Although the predominant species of the above three sphingolipid classes were consistent in chloroplasts and TL, the most abundant species of GlcCer showed a clear difference between the chloroplast and TL samples.In TL, the t18:1 h24:1 form was the predominant species, while in chloroplast samples, t18:0 h16:0 and d18:1 h16:0 forms were the predominant species (Figure 6c).This further underscores the specificity of chloroplast sphingolipid composition.

Discussion
This study presents the detailed identification of the sphingolipid composition of Arabidopsis chloroplasts.The sphingolipidome of the total leaves (TL) identified in our study aligns with those reported in previous studies [14,28].When compared to other reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.reported sphingolipidomes in plant tissues and cellular structures, the chloroplast exhibited commonalities and unique characteristics.
In terms of sphingolipid class distribution, GIPCs constituted the primary sphingolipid class in chloroplasts, accounting for approximately 60% of the total.This is similar to observations for the plasma membrane (PM), detergent-resistant membrane (DRM), microsomal membrane (MIC), endoplasmic reticulum (ER), nuclei, and mitochondria (Mito) [22][23][24].GIPCs have been previously reported to serve as crucial membrane components in plants for pathogen recognition [29], as well as in response to environmental stresses such as elevated levels under cold stress and assisting in the influx of Ca 2+ under salt stress [30,31].The structural complexity and predominance of GIPCs could align with a wide variety of membrane functions, although the exact functional role and mechanism of GIPCs in chloroplasts remain unknown.Moreover, the proportion of GIPCs to the total sphingolipids in chloroplasts was lower than that in TL, while there was a more pronounced presence of free LCBs and hCer in the chloroplast.
Robust lipid trafficking between the chloroplast and ER occurs within the endomembrane system [32,33], suggesting that the accumulation of free LCBs in the chloroplast could function as a reservoir for sphingolipid biosynthesis.Notably, the LCB has been reported to be a potent signaling molecule that influences phytohormone levels and plays pivotal roles in programmed cell death [34,35], hinting at a potential relationship between the accumulation of LCBs in chloroplasts and the execution of these functions.As for hCer, their abundant presence in chloroplasts could be attributed to their hydrophobic properties.It has been reported that the stratum corneum necessitates the hydrophobicity of hCers to regulate water flow [36], and this property of hCers has been utilized to elucidate the accumulation of hCers in vacuolar membranes (VMs) [23], suggesting that the accumulation of hCers in chloroplasts may be driven by a similar mechanism.
For specific sphingolipid composition, the individual characteristics among TL and other cellular structures warrant further attention.To achieve this purpose, we compared the results from previous studies [22][23][24] with our results for a comprehensive discussion (Tables 1 and 2).It is noteworthy that all reported sphingolipid data are derived from LC-MS/MS technology.To ensure consistency in their comparison, original data have been converted to relative content.For Cer, the primary components remained consistent across the reported cellular structures, with t18:1 c24:0 and t18:1 c26:0 predominating, except for Mito, where d18:1 c24:1 was the most abundant component (Table 2).Additionally, a higher presence of 16C forms (primarily d18:1 c16:0) was observed in MIC, VM, chloroplast, and PM compared to other sample types (Table 1).
For hCer, t18:1 h24:0/1 were the predominant forms across each cellular structure and in the TL, with Mito also exhibiting a higher presence of d18:1 h24:0 (Table 2).Interestingly, d18:1 h24:0 also appeared as an abundant fraction in the TL (Figure 6a), suggesting that this species could be significantly contributed by Mito.Additionally, the nuclei were characterized by a substantial accumulation of sphingolipid with the t18:0 form and 26C form, distinguishing it from other structures (Table 1).
Previous reports have indicated that very long-chain fatty acids (VLCFAs) (including 24C and 26C) are the primary sphingolipid components and play a crucial role in plant growth and development [39,40].However, the results presented above suggest that internal cellular structures, such as the PM, chloroplast, and VM, exhibit a significant propensity to accumulate sphingolipids with 16C forms.Previous in vitro studies discovered that the connections between VLCFA ceramides exhibit stronger interactions, facilitating the establishment of stable and complex biological systems.In contrast, shorter 16C fatty acids are mostly phase-separated [41].Therefore, the accumulation of the 16C form in the PM, chloroplast, and VM could potentially be associated with the motility of the endomembrane system and its intricate metabolic processes.Interestingly, the sphingolipid composition of Mito appears to be unusual in that it contains a large number of complex sphingolipids in the form of d18:1 and 24C, and sphingolipids with the d18:2 form and 28C form were identified in Mito [24], but not detected in the chloroplast or in other reported cellular structures.Historically, d18:2 has only been found in the pollen and flowers of Arabidopsis [42].It remains unclear whether these components are related to the specific function of Mito.These findings further suggest that the distribution of sphingolipids is asymmetrical and uneven in plant cells, and whether this distinction in sphingolipid classes and species correlates with organelle-specific functions warrants further exploration.
Furthermore, it should be noted that in this study, the entire chloroplast was extracted without distinguishing specific structures, such as the inner membrane, outer membrane, thylakoids, and stroma.Therefore, the distribution of sphingolipids within each part of the chloroplast remains unknown.
In conclusion, our study pioneers the examination of the sphingolipidome of plant chloroplasts, thereby enhancing the sphingolipid profile of plant organelles and providing a robust reference for comprehending the spatial distribution and functional roles of sphingolipids across different cellular structures.Simultaneously, gaining insights into the sphingolipid composition of chloroplasts, which constitute a significant pool of organic carbon sources in plants, is of paramount importance for a profound understanding of chloroplast functions.

Plants and Growth Conditions
Arabidopsis thaliana wild-type (Columbia, Col-0) seeds were sterilized with chlorine for 1h and then spread on ½ of the MS medium.After 3 d of stratification at 4 • C, the seeds were grown at 22 • C under a 16 h light/8 h dark cycle with a 4800 to 6000 lux light intensity for 1 week before planting in a soil mixture (a mix of peat soil and vermiculite at 3:1, v:v).All the leaves were harvested at 4 weeks old.Some leaves were used for chloroplast isolation immediately, and others were lyophilized and then stored at −80 • C

Figure 1 .
Figure 1.Experimental procedure for the sphingolipid analysis and purity assessment.(a) An experimental approach to obtain total leaf (TL) and chloroplast samples from Arabidopsis plants for sphingolipid analysis.Yellow asterisks indicate a target precipitation containing chloroplasts.(b)Immunodetection of BAK1 from the plasma membrane, BIP2 from the endoplasmic reticulum, RbcL from chloroplasts, V-ATPase from vacuoles, VDAC1 from the mitochondria, and histone H3 from nuclei.Proteins were separated using SDS-PAGE and detected via immunoblot.At least two biological replicates were performed for immunodetection with all protein markers.RbcL was used as a loading control.

Figure 2 .
Figure 2. Multivariate analysis and heatmap plots of sphingolipid profiles.(a) Representation of the structure of main complex sphingolipid classes.The R1 in the structure of GIPC indicates mannose, glucosamine, N-acetyglucosamine, galactose, arabinose, or a combination of these hexose groups as described in [27].(b) Partial least squares-discriminant analysis (PLS-DA) of sphingolipid profiles of the chloroplast and total leaf samples.The analysis is based on the relative content (mol %) of all detected sphingolipid species.In this study, nine individual plants were set for each sample type.(c)Heatmap plots of the abundance of LCBs, Cers, hCers, GlcCers, and GIPCs.The Z-score represents the standardized content of individual sphingolipid species in mol %.The scale from the left value to the right value represents the number of standard deviations from the average of each row (i.e., sample types), with red indicating higher contents than average and the blue color indicating lower contents than average.An "O" in the heatmap means that sphingolipid species were not detected in this sample group.The sphingolipid species that were not detected in any of the samples are not shown.

Figure 2 .
Figure 2. Multivariate analysis and heatmap plots of sphingolipid profiles.(a) Representation of the structure of main complex sphingolipid classes.The R1 in the structure of GIPC indicates mannose, glucosamine, N-acetyglucosamine, galactose, arabinose, or a combination of these hexose groups as described in [27].(b) Partial least squares-discriminant analysis (PLS-DA) of sphingolipid profiles of the chloroplast and total leaf samples.The analysis is based on the relative content (mol %) of all detected sphingolipid species.In this study, nine individual plants were set for each sample type.(c)Heatmap plots of the abundance of LCBs, Cers, hCers, GlcCers, and GIPCs.The Z-score represents the standardized content of individual sphingolipid species in mol %.The scale from the left value to the right value represents the number of standard deviations from the average of each row (i.e., sample types), with red indicating higher contents than average and the blue color indicating lower contents than average.An "O" in the heatmap means that sphingolipid species were not detected in this sample group.The sphingolipid species that were not detected in any of the samples are not shown.

Figure 3 .
Figure 3. Content and distribution of sphingolipid classes.(a) Absolute content of five s classes (LCBs, Cers, hCers, GlcCers, and GIPCs).The sphingolipid contents were expres of the protein in chloroplasts.Error bars represent the means ± SE from nine individua Distribution of sphingolipid classes.Percentage values in pie charts represent the aver content of each sphingolipid class in chloroplast and total leaf samples.

Figure 3 .
Figure 3. Content and distribution of sphingolipid classes.(a) Absolute content of five sphingolipid classes (LCBs, Cers, hCers, GlcCers, and GIPCs).The sphingolipid contents were expressed per mg of the protein in chloroplasts.Error bars represent the means ± SE from nine individual plants.(b) Distribution of sphingolipid classes.Percentage values in pie charts represent the average relative content of each sphingolipid class in chloroplast and total leaf samples.
TL samples, indicating that chloroplasts have a unique sphingolipid composition.

Figure 6 .
Figure 6.Distribution of abundant species from four sphingolipid classes.(a) Cers, (b) hCers, (c) GlcCers, and (d) GIPCs.The size of each bubble is related to the average relative content from nine individual plants for each individual species.Only sphingolipid species with an abundance of more than 1% for each sphingolipid class (16 species of Cers, 14 species of hCers, 9 species of GlcCers, and 14 species of GIPCs) are shown.Different letters labeled on the top of bars indicate statistical differences using two-tailed Student's t-tests (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001).

Figure 6 .
Figure 6.Distribution of abundant species from four sphingolipid classes.(a) Cers, (b) hCers, (c) GlcCers, and (d) GIPCs.The size of each bubble is related to the average relative content from nine individual plants for each individual species.Only sphingolipid species with an abundance of more than 1% for each sphingolipid class (16 species of Cers, 14 species of hCers, 9 species of GlcCers, and 14 species of GIPCs) are shown.Different letters labeled on the top of bars indicate statistical differences using two-tailed Student's t-tests (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001).

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.
TL Plants 2024, 13, x FOR PEER REVIEW 9 of 15

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 1 .
Sphingolipid composition of cellular structures in Arabidopsis thaliana.

Table 2 .
Predominant sphingolipid species of cellular structures in Arabidopsis thaliana.