Multiomic analyses of two sorghum cultivars reveals the change of membrane lipids in their responses to water deficit

https://doi.org/10.1016/j.plaphy.2022.02.015Get rights and content

Highlights

  • Drought decreased the unsaturation indices of monogalactosyl diglyceride and phosphatidylglycerol and phosphatidylcholine.

  • The digalactosyl-/monogalactosyl-diacylglycerol ratio increased in drought-sensitive cultivar under drought condition.

  • Responses of membrane lipids to drought depended on cultivars.

  • Eleven candidate genes involved in membrane lipid metabolism were obtained in sorghum.

Abstract

Drought is one of the main abiotic stresses influencing crop production all over the world. Membranes are sensitive to drought stress and easy to be degraded and modified. Lipidome and transcriptome analyses were applied to analyze the responses of membrane lipids to drought stress in two sorghum (Sorghum bicolor (L.) Moench) cultivars, drought-sensitive cv. Hongyingzi and drought-tolerant cv. Kangsi. In total, 156 lipid compounds were identified and the contents of the predominant ones changed significantly under drought stress. Drought significantly decreased the unsaturation indices (UI) of digalactosyl-diacylglycerol (DGDG), monogalactosyl-diacylglycerol (MGDG), phosphatidylglycerol (PG) and phosphatidylcholine (PC) in both cultivars, except for insignificant changes of UI for DGDG in cv. Kangsi. Transcriptome sequencing analysis identified genes related to membrane lipid remodeling such as phospholipase D α1 (PLDα1), phospholipase D δ (PLDδ), and phospholipase A 2 (PLA2). By integrating transcriptome data and lipidome data, weighted gene co-expression network analysis (WGCNA) identified hub genes, transcription factors and the genes involved in lipid metabolism. Then, the protein and protein interaction (PPI) was analyzed using STRING and the possible candidate genes regulating membrane lipids under drought stress were obtained, including CCT2, CER1, DGK1, DGK5, EMB3174, KCS4, LCB2, PAH1, PLDP1, PKP-β1, and KCS11. The results from this study have the potential to accelerate the process to breed drought-tolerant sorghum lines.

Introduction

Drought is considered as one of the major abiotic stresses influencing crop production all over the world (Levitt, 1980). Plants defend against drought stress mainly through four mechanisms, avoidance, escape, tolerance, and drought recovery (Fang and Xiong, 2015), among which tolerance and drought recovery are regarded as the two important strategies (Tari et al., 2013). Drought stress is first perceived by plant membranes, and degradative processes and modifications in membrane lipid compositions have been reported in numerous plant species subjected to drought stresses (Gigon et al., 2004; Pham-Thi et al., 1985). During dehydration, lipid remodeling is one of the critical mechanisms to keep cell membrane integrity. Generally, a decrease in the contents of polar membrane lipids is observed in drought-stressed plants (Benhassaine et al., 2002), and the membrane integrity and stability of plants during drought are key indicators reflecting plant drought tolerance (Gasulla et al., 2013; Su et al., 2009).

The structural components of photosynthetic membranes mainly include galactosylglycerides, monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG), as well as minor amounts of phospholipids, mostly phosphatidylglycerol (PG) (Monteiro et al., 1990). In order to adapt to the environmental changes, plants would accumulate more DGDG and MGDG to regulate membrane stability and integrity of thylakoid for photosynthetic electron transport (Chen et al., 2018a; Cowan, 2006; Thole and Nielsen, 2008). Under drought conditions, some signaling lipids, such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositols (PIs) (Cowan, 2006), are significantly accumulated to activate downstream signaling pathways, which finally leads to physiological responses (Testerink and Munnik, 2011). Drought stress generally results in a decrease of MGDG, causing an increase of the DGDG/MGDG ratio (Vrablik and Watts, 2012). A decrease in fatty acid unsaturation level is also observed in some drought-stressed plants, which increases the membrane fluidity (Liu et al., 2019). Studies have shown that adaptation to dehydration involves membrane lipid remodeling and fatty acid desaturation (Okazaki and Saito, 2014), and the fluidity of the lipid membrane is determined by the unsaturated fatty acids (Kachroo and Kachroo, 2009). Based on a meta-analysis (Dawid et al., 2016), drought stress tends to increase the levels of 16:0 fatty acid and 18:2 fatty acid while reducing the levels of 16:1 fatty acid, 16:3 fatty acid and 18:3 fatty acid, which improved membrane fluidity and stability and plant tolerance to drought stress (Narayanan et al., 2016). A modification of the degree of lipid order in Medicago truncatula is also observed in drought susceptible genotype, which could be proposed as mechanisms required for signal transduction (Couchoud et al., 2019). Therefore, membrane lipid compositions may provide reliable parameters in breeding or selecting drought-tolerant crop cultivars for arid environments (Ohlrogge, 1994).

The biosynthesis pathways of membrane lipids start from C16 and C18 fatty acids synthesized in plastids. Then, the fatty acids are transported via the chloroplast inner envelope to the endoplasmic reticulum (ER), where they are used for glycerolipid synthesis, with phosphatidic acids (PA) as initial products and PG, MGDG, DGDG, sphingolipids (SL), PC, PE, and PI as final products (Mullet et al., 2005). Fatty acid biosynthesis in plants involves a very complicated biological pathway. Fatty acid desaturases (FADs) are key enzymes that introduce double bonds into fatty acids during the synthesis of glycerolipids (Zhong et al., 2011), such as FAD2, FAD3, FAD4, FAD5, FAD6, FAD7 and FAD8 (Wang et al., 2019). Among them, FAD2 and FAD6 introduce double bonds at the omega-6 (Δ-12) position to catalyze oleic acid (18:1) to linoleic acid (18:2). FAD3, FAD7 and FAD8 are the key enzymes that introduce double bonds at the omega-3 (Δ-15) position to catalyze the conversion of 16:2 or 18:2 into 16:3 or 18:3, respectively (Anders and Huber, 2010; Wang et al., 2019). DGD1, a gene encoding a galactosyltransferase-like protein, involves in the assembly of the thylakoid lipid matrix and subcellular lipid trafficking in Arabidopsis thaliana (Dörmann et al., 1999). SENSITIVE TO FREEZING 2, a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane (Moellering et al., 2010). With the isolation of more genes involved in lipid biosynthesis, we can improve plant drought tolerance through genetic engineering.

RNA sequencing (RNA-Seq) is a rapid and convenient method to find key genes in fatty acid biosynthesis and unsaturated fatty acid biosynthesis at the molecular level (Huang et al., 2021). The integration of metabolic changes with transcriptome data has been reported in discovering genes related to secondary metabolites (Saito et al., 2008). For example, an integrated lipidomic and transcriptomic study suggested that Arabidopsis transcriptionally coordinated the regulation of glycerolipid metabolism under environmental stresses, and stress reduced the expression of transcription factors and genes encoding enzymes and proteins involved in lipid trafficking (Higashi et al., 2015; Szymanski et al., 2014). Transient heat stress misregulated lipid biosynthesis-related genes, resulted in increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids in maize (Begcy et al., 2019). Under drought, oak plants inoculated with mycorrhizal fungi showed increased expression of genes involved in neutral lipids biosynthesis, increasing plant abilities to tolerate drought stress (Sebastiana et al., 2019).

Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal all over the world and is widely distributed in tropical and subtropical areas (Sanjari et al., 2021). Though sorghum is one of the most drought-tolerant grain crops (Wang et al., 2020), its yield still reduces significantly under drought stresses (Sanchez et al., 2002). At present, the molecular mechanisms of the membrane lipid biosynthesis under drought stress have been characterized in Arabidopsis and some other plant species, however, the molecular mechanism and the genes related to membrane lipid biosynthesis remain largely unknown for sorghum. Recently, a study on sorghum has shown that transcriptomics revealed chilling-induced up-regulation of cold response transcription factor and genes including lipid remodeling phospholipase D alpha 1 gene in the chilling-tolerant Chinese line (Marla et al., 2017). In our previous study, integrative analysis of the cuticular lipidome and transcriptome of S. bicolor revealed cultivar variance in drought tolerance (Zhang et al., 2021). In the present study, the two sorghum (Sorghum bicolor) cultivars with potentially different drought resistance, cv. Kangsi and cv. Hongyingzi, were used to analyze the chemical profiles of membrane lipids in drought-stressed seedlings, and transcriptome sequencing (RNA-seq) was also applied to investigate the transcriptional regulation of the lipid biosynthesis machinery. A weighted gene co-expression network analysis (WGCNA) network was constructed by integrating transcriptome data and lipidome data. The main objectives of this study were (a) to analyze the chemical profiles of sorghum membrane lipids, (b) to explore the genes involved in lipid biosynthesis under drought-stressed conditions, and (c) to elucidate the mechanisms of sorghum cultivars in their different responses to drought stress.

Section snippets

Plant material and drought stress treatment

The seeds of the sorghum cv. Hongyingzi and cv. Kangsi were bought from the Hanqing Seed Company of Hebei, China. Seeds were sterilized with 10% H2O2 for 10 min, rinsed three times with distilled water, and then sowed in pots (10 cm in diameter and 15 cm in depth) filled with a mixture of peat, soil and vermiculite (2/3/1, V/V/V). The pots were transferred to growth chambers (RXZ-500D, Ningbo southeast instrument co. LTD) controlled at 28 °C/25 °C day/night temperatures with 80% relative

Response of lipidome to drought stress

In total, 156 lipid compounds were detected from leaves of well-watered and drought-stressed sorghums (Table S2), including three plastidic lipids, digalctosyldiacul glycerol (DGDG), monogalactosyl diacylglycerol (MGDG) and phosphatidylglycerol (PG); five extraplastidic phospholipids, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS); and three lysolipids, lysophosphatidylglycerol (LPG), lysophosphatidylcholine

Discussion

MGDG and DGDG are the main components of the lipid bilayer matrix of chloroplast envelopes and thylakoid membranes, playing important roles in plant tolerance to abiotic stresses (Gigon et al., 2004; Shimojima et al., 2015). DGDG forms stable bilayer lamellar phase whereas MGDG is more likely to form an unstable hexagonal micelle in chloroplast membranes (Jouhet, 2013; Shipley et al., 1973). Studies have shown that plants tend to increase DGDG/MGDG ratio to maintain stability of the chloroplast

Conclusion

Lipidomic and transcriptomic analyses were applied to analyze the responses of membrane lipids to drought stress in two sorghum cultivars. Total of 156 lipid compounds were identified. The contents of MGDG, DGDG, PG and PE decreased and PA increased in both sorghum cultivars under drought-stressed conditions, PC, PI and PS decreased significantly in cv. Hongyingzi but not in cv. Kangsi; higher DGDG/MGDG ratio was found in drought-sensitive cv. Hongyingzi; drought also decreased the UI of DGDG,

Author contribution

DX and XZ carried out the experiments and data analysis. DX, YN and YG interpreted the results and wrote the manuscript. All authors provided critical feedback, helped shape the research and authorized the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was funded by National Natural Science Foundation of China (31670407) and First Class Grassland Science Discipline” program in Shandong Province, China.

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