Chilling temperature remodels phospholipidome of Zea mays seeds during imbibition

Abstract

Global warming is a major agricultural issue in the Northern hemisphere where higher temperatures are expected to be associated with restricted water availability. In Europe, for maize, earlier and further northward sowings are forecasted in order to avoid water deficit periods in the crop life cycle. However these conditions may compromise seed germination and stand establishment since they will take place at cold temperatures. It is urgent to better understand the molecular bases of response of germinating maize seeds to cold in order to design genotypes adapted to these novel agricultural practices. Here we have performed a global phospholipidomic study to profile changes in membrane reorganisation during seed imbibition at 10 °C of cold-tolerant and -sensitive maize hybrids. Using a Multiple Reaction Monitoring (MRM-MS/MS) method coupled with HPLC we have identified 80 distinct phospholipids. We show that seed sensitivity to cold temperatures during imbibition relies on the accumulation of saturated or poorly unsaturated fatty acids, whatever the phospholipid class. In contrast seeds of cold-tolerant hybrid accumulated polyunsaturated chains which was associated with lower electrolyte leakage during imbibition at 10 °C. The expression of fatty acid desaturase genes provides a molecular model of maize seed sensitivity to imbibitional chilling damage.

Figure 1

Effect of temperature on seed germination. Germination of hybrids A (a) and B (a) at 4 different temperatures and cardinal temperatures for germination (c). Each point is the mean of 3 replicates of 25 seeds each ± SD.

Figure 2

Malondialdehyde (MDA) embryo content (a) and electrolyte leakage (b) of seeds of hybrids A and B after 24 h imbibition at 10 °C and 18 °C. Means of 3 replicates. Vertical bars correspond to SD. Asterisks above the bar indicate that electrolyte leakage in hybrid B at 10 °C differed significantly from that measured at 18 °C (*P < 0.05).

Figure 3

Phospholipid profiling of seeds of hybrids A (a) and B (b). Molecular species composition of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) extracted from embryos of hybrids A. Composition of fatty acid couples is expressed in percentage of total molecular species in each phospholipid class. Seeds were either dry (black bars) or imbibed at 10 °C (shaded bars) or 18 °C (white bars). Values are means ± SD of 3 triplicates.

Figure 4

Principal component analysis (PCA) of membrane composition of embryos of hybrids (A and B). Score plot of lipid species content of dry seeds (green) and seeds imbibed for 24 h at 10 °C (blue) or 18 °C (red). Each letter represents a replicate of hybrid (A or B). Principal components 1 and 2 (PC1 and PC2) explain 38.16% and 15.68% of the variance in the data set. Loadings can be found in Supplemental Table S2.

Figure 5

K-mean clustering analysis of lipid species after 24 h imbibition at 10 °C or 18 °C. Each case represents the ratio between phospholipid composition of imbibed to dry embryos. Red is used for ratios < 1, green for ratios > 1 and white for ratio = 1. Clusters 1 and 2 cluster species with reduced or increased ratio in hybrid B when imbibed at 10 °C, respectively.

Figure 6

Fatty acid composition in phosphatidylcholine (PC). Proportion (in %) of fatty acid chains in dry and imbibed embryos of hybrids A and B. DBI: double bond index, ACL: acyl carbon length. Different letters denote significantly different mean values at P < 0.05 according to Tukey’s multiple range test. Absence of letters indicates that means are not significantly different.

Figure 7

Transcript abundance of desaturase transcripts in maize embryos. Expression of ssi2 (a), fad2 (b) and fad6 (c) in dry embryos of hybrid A (black bars) and hybrid B (white bars) and various durations of imbibition at 10 °C or 18 °C. The relative expression was calculated from qRT-PCR data with 3 references genes, cdk, 2og-fe and unknown, and expressed in arbitrary units with a value of 100 attributed to the dry seeds. Means ± SD of three biological replicates are shown.