Two Triacylglycerol Lipases Are Negative Regulators of Chilling Stress Tolerance in Arabidopsis

Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology.


Introduction
Cold stress significantly limits plant growth and development and causes substantial yield losses in crops worldwide. Cold stress can be experienced by plants as chilling stress and freezing stress. Chilling stress is critical for tropical and subtropical plants including tomato, soybean, sweet potato, and eggplant because they are not adapted to unpredicted sudden temperature drops (i.e., unforeseen frosts). Very little is known about the molecular nature of chilling stress responses in plants, whereas the molecular mechanisms of freezing stress tolerance in plants have been extensively studied. Cold temperature rapidly induces the expression of a small group of Apetala2 (AP2)/ethylene response factor (ERF)-type transcription factors termed dehydration responsive element (DRE)/C-repeat (CRT)-binding factors (CBFs/DREBs), which in turn control the expression of cold-responsive (COR) genes through binding to the DRE/CRT cis-element (consensus sequence of CCGAC) in the COR gene promoter regions [1][2][3]. The inducer of CBF expression 1 (ICE1), which is a basic helix-loop-helix transcription factor, functions as an upstream regulator of CBFs by binding to the MYC recognition sequences in the CBF3 gene promoter and induces its expression under cold stress [4]. The ICE1 itself is subject to posttranslational modifications including phosphorylation, ubiquitination, and sumoylation under cold stress. A RING finger protein, HOS1, acts as an E3 ligase to mediate the polyubiquitination of ICE1 for degradation by the 26S protease complex under cold stress. Phosphorylation of ICE1 is controlled by multiple protein kinases. Open stomata 1 (OST1), a serine/threonine protein kinase involved in ABA signaling, phosphorylates ICE1 and suppresses the HOS1-mediated ICE1 degradation under cold stress [5]. Thus, OST1 functions as a positive regulator of the ICE1-CBF3 pathway for freezing tolerance. A recent study showed that two mitogen-activated protein kinases, MPK3 and MPK6, phosphorylate ICE1 and promote its degradation under cold stress to attenuate the ICE1-CBF3 pathway, while two calcium/calmodulin-regulated receptor-like kinases (CRLK1 and CRLK2) and MPK4 negatively regulate the kinase activities of MPK3 and MPK6 to allow the accumulation of ICE1 under cold stress [6]. It had been shown that SIZ1, a SUMO E3 ligase, facilitates sumoylation of ICE1 and that this posttranslational modification may activate and/or stabilize ICE1 to permit CBF3 gene expression, leading to freezing tolerance [7].
Triacylglycerols (TAGs), three fatty acids esterified to glycerol, are major storage reserves of carbon and energy for oil seeds, pollens, and fruits, and they constitute up to 60% of the dry mass of oil seeds, while TAGs only make up a small percentage of the total lipids TAGs in vegetative tissues such as leaves and roots. TAG lipases de-esterify fatty acids from TAGs at each of the sn1, sn2, and sn3 positions. The active site of a TAG lipase consists of three amino acids (Ser, Asp or Glu, and His). TAG lipases can also hydrolyze diacylglycerols (DAGs) and other substrates including monoacylglycerol (MAGs), glycerol(phospho)lipids in position sn1, or sterol esters [8][9][10]. Therefore, TAGs are the major sources of fatty acids, which can be further metabolized through β-oxidation in peroxisomes to yield acetyl CoA, a key metabolite for energy production in mitochondrial respiration and for the synthesis of carbohydrates through the glyoxylate cycle and gluconeogenesis during seed germination and early seedling establishment. They are also critical for the normal growth and development of adult plants. DAGs can be used for the assembly of membrane lipids. TAG turnover by TAG lipases also accounts for the low accumulation of TAGs in vegetative tissues [11,12]. Characterization of a plastid TAG lipase from Arabidopsis revealed that TAG lipase is important in maintaining the structural integrity of chloroplasts, possibly by mobilizing the fatty acids of plastoglobular TAG [13]. It had been shown that the Arabidopsis MPL1 (MYZUS PERSICAE-INDUCED LIPASE1; encoded by At5g14180) has lipase activity and that it plays an important role against the green peach aphid [14]. The Arabidopsis LIP1 (encoded by At2g15230) has TAG lipase activity and lip1 seedlings displayed no altered growth rates under normal growth conditions, but the lip1 mutant seedlings accumulated more TAG than the wild type [15].
In this study, we aimed to determine the role of TAG lipases in cold stress responses through detailed characterization of the Arabidopsis mpl1 and lip1 single mutants and the double-mutant mpl1lip1. Our results indicate that MPL1 and LIP1 are partially redundant in cold stress responses and that both are negative regulators of chilling stress tolerance.

Phylogenetic Analysis of MPL1 and LIP1, Expression Profiles of MPL1 and LIP1, and Generation of mpl1lip1 Double Mutant
To assess the function of triacylglycerol lipases in cold stress responses, we examined expression profiles of two triacylglycerol lipases from Arabidopsis thaliana, MPL1 and LIP1. MPL1 and LIP1 share high sequence homologies with triacylglycerol lipases from other plant species (Figure 1). LIP1 is expressed in all plant tissues and reaches peak expression level in senescing leaves (Figure 2A), while MPL1 is expressed at relatively lower levels than MPL1 and its peak expression is found in the mature pollens ( Figure 2B). The expression of LIP1 is downregulated by cold stress in the root tissues ( Figure 2C) or in whole seedlings ( Figure 3E). The expression of MPL1 is downregulated by cold stress in the shoot tissues ( Figure 2D) or in whole seedlings ( Figure 3D). These results suggest that MPL1 and LIP1 might act as negative regulators of cold stress. Essentially LIP1 is not responsive to salt stress or heat stress ( Figure 2E,G). MPL1 is moderately upregulated in roots or downregulated in shoots under salt stress ( Figure 2F). MPL1 is also downregulated in shoots under heat stress ( Figure 2H). These results suggest that MPL1 may play a role in salt stress or heat stress.
We obtained two T-DNA insertional mutants for MPL1 (SALK_101919; mpl1) and LIP1 (SALK_114605; lip1) and identified homozygous mutant plants through genotyping ( Figure 3A). The expression of MPL1 was significantly reduced in the mpl1 seedlings, and the expression of LIP1 was almost undetectable in the lip1 seedlings ( Figure 3B,C). The expression of MPL1 in the lip1 seedlings and the expression of the LIP1 in the mpl1 seedlings were significantly reduced, suggesting that there is a feedback regulation of MPL1 by the lip1 mutation and there is a feedback regulation of LIP1 by the mpl1 mutation ( Figure 3B,C). To overcome potential functional redundance of MPL1 and LIP1, we generated the mpl1lip1 double mutant by crossing the mpl1 and lip1 single-mutant plants. The expression of the MPL1 and LIP1 was significantly low in the mpl1lip1 double-mutant plants ( Figure 3B,C).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW expression of LIP1 is downregulated by cold stress in the root tissues ( Figure 2C whole seedlings ( Figure 3E). The expression of MPL1 is downregulated by cold st the shoot tissues ( Figure 2D) or in whole seedlings ( Figure 3D). These results sugge MPL1 and LIP1 might act as negative regulators of cold stress. Essentially LIP1 is sponsive to salt stress or heat stress ( Figure 2E,G). MPL1 is moderately upregula roots or downregulated in shoots under salt stress ( Figure 2F). MPL1 is also dow lated in shoots under heat stress ( Figure 2H). These results suggest that MPL1 may role in salt stress or heat stress.   in different organs and developmental stages of wild-type plants as determined by microarrays [16]. (C,D) Expression profiles of LIP1 and MPL1 under cold stress as determined by microarrays with 13-day-old wild-type seedlings subjected to cold treatments [17]. (E,F) Expression profiles of LIP1 and MPL1 under salt stress as determined by microarrays with 13-day-old wild-type seedlings subjected to cold treatments [17]. (G,H) Expression profiles of LIP1 and MPL1 under salt stress as determined by microarrays with 16-day-old wild-type seedlings subjected to heat stress at 38 °C for 0, 0.25 (+0 h recovery at 25 °C), 0.5 (+0 h recovery at 25 °C), 1 (+0 h recovery at 25 °C), or 3 h (+3 h recovery at 25 °C = 6 h; +9 h recovery at 25 °C = 12 h; +21 h recovery at 25 °C = 24 h) [17]. Data are means ± sd (n = 3).

A B
Relative transcript level

24
Root Shoot
We obtained two T-DNA insertional mutants for MPL1 (SALK_101919; mpl1) and LIP1 (SALK_114605; lip1) and identified homozygous mutant plants through genotyping ( Figure 3A). The expression of MPL1 was significantly reduced in the mpl1 seedlings, and the expression of LIP1 was almost undetectable in the lip1 seedlings ( Figure 3B,C). The expression of MPL1 in the lip1 seedlings and the expression of the LIP1 in the mpl1 seedlings were significantly reduced, suggesting that there is a feedback regulation of MPL1 by the lip1 mutation and there is a feedback regulation of LIP1 by the mpl1 mutation (Figure 3B,C). To overcome potential functional redundance of MPL1 and LIP1, we generated the mpl1lip1 double mutant by crossing the mpl1 and lip1 single-mutant plants. The expression of the MPL1 and LIP1 was significantly low in the mpl1lip1 double-mutant plants ( Figure 3B,C).

The mpl1, lip1, and mpl1lip1 Plants Display Increased Chilling Stress Tolerance at Seed Germination and Post-Germination Seedling Development Stages
Frost in late fall or early spring can be a limiting factor for seed germination of temperate plant species. We simulated the frost conditions by incubating MS agar plates with freshly sown seeds at chilling temperature in a growth chamber. It took about several weeks for the seeds to germinate, and approximately two additional weeks for the seedlings with fully expanded cotyledons and the first leaf to emerge. Under control conditions, there were no morphological differences among the mpl1, lip1, mpl1lip1, and Col-0 seedlings and the mpl1lip1 seedlings accumulated slightly more chlorophyll than the Col-0 seedlings ( Figure 4A,B). Under chilling stress, the mpl1 and lip1 plants were moderately chilling tolerant as indicated by more green seedlings and increased accumulation of  Data are means ± sd (n = 4). One-way ANOVA (Tukey test) was performed, and significant difference is indicated by different lowercase letters (p < 0.05).

The mpl1, lip1, and mpl1lip1 Plants Display Increased Chilling Stress Tolerance at Seed Germination and Post-Germination Seedling Development Stages
Frost in late fall or early spring can be a limiting factor for seed germination of temperate plant species. We simulated the frost conditions by incubating MS agar plates with freshly sown seeds at chilling temperature in a growth chamber. It took about several weeks for the seeds to germinate, and approximately two additional weeks for the seedlings with fully expanded cotyledons and the first leaf to emerge. Under control conditions, there were no morphological differences among the mpl1, lip1, mpl1lip1, and Col-0 seedlings and the mpl1lip1 seedlings accumulated slightly more chlorophyll than the Col-0 seedlings ( Figure 4A,B). Under chilling stress, the mpl1 and lip1 plants were moderately chilling tolerant as indicated by more green seedlings and increased accumulation of chlorophyll (Figure 4 A,C,D). The mpl1lip1 plants showed additive effect of the mpl1 and lip1 mutationsthat is the mpl1lip1 plants were chilling tolerant with proportionally more green seedlings and increased accumulation of chlorophylls than the mpl1 and lip1 single mutants under chilling stress (Figure 4 A,C,D). These results suggest that MPL1 and LIP1 are negative regulators of chilling stress tolerance.
Below are the updated Figures 4, 5, 6 and 7

The mpl1, lip1 and mpl1lip1 Plants Are Chilling Tolerant When Grown in the Dark
To determine whether the increased chilling stress tolerance in the mpl1, lip1, and mpl1lip1 plants depends on the developmental stages, we examined the response of the mpl1, lip1, and mpl1lip1 seedlings to chilling temperature in the dark after their seeds had germinated. We measured the hypocotyl elongation as an indicator of chilling stress responses. Essentially there were no differences in hypocotyl elongation among Col-0, mpl1, lip1, and mpl1lip1 seedlings under control conditions ( Figure 5A,B). Under chilling stress, the mpl1, lip1, and mpl1lip1 seedlings developed significantly longer hypocotyls than the Col-0 seedlings, indicating that they were chilling tolerant ( Figure 5A,C).  (C) Hypocotyl elongation of Col-0, mpl1, lip1, and mpl1lip1 under chilling stress for 42 days. Data are means ± sd (n = 4 in (B) 12 in (C) (n indicates the number of MS medium plates)). One-way ANOVA (Tukey test) was performed, and significant difference is indicated by different lowercase letters (p < 0.03).

The mpl1, lip1, and mpl1lip1 Mutations Do Not Alter the Responses to Freezing Temperatures or Heat Stress
We speculate that the mpl1, lip1, and mpl1lip1 mutations may cause additional phenotypic alterations to other abiotic stress conditions. In addition to chilling stress, freezing stress is the second component of the cold stress. We examined the responses of the mpl1, lip1, and mpl1lip1 plants to freezing temperatures with detached rosette leaves from soil-grown plants. Without cold acclimation, the mpl1, lip1, and mpl1lip1 plants released similar amounts of electrolytes from damaged biological membranes caused by freezing temperatures as the Col-0 plants ( Figure 6A). After cold acclimation at 4 • C for one week, the mpl1, lip1, and mpl1lip1 plants released similar amounts of electrolytes from damaged biological membranes caused by freezing temperatures to the Col-0 plants ( Figure 6A). These results indicate that the mpl1, lip1, and mpl1lip1 mutations do not alter the responses to freezing temperatures.  We subsequently examined the responses of the mpl1, lip1, and mpl1lip1 plants to another temperature extreme, heat. We used hypocotyl elongation of heat-treated seedlings that grew in the dark as an indicator for heat stress responses. All plants tested displayed similar length of hypocotyls under control conditions, and they had similarly reduced length of hypocotyls by one-hour treatment at 45 • C ( Figure 6B). These data indicate that the mpl1, lip1, and mpl1lip1 mutations do not alter the responses to heat stress.

The mpl1, lip1, and mpl1lip1 Mutations Alter Gene Expression under Chilling Stress
We performed RNA-seq experiments to determine whether the mpl1, lip1, and mpl1lip1 mutations affect gene expression (Supplemental Table S1). Data analysis of the RNA-seq results revealed that the mpl1 mutation had a modest effect on gene expression under both control and chilling stress conditions ( Figure 7A and Supplemental Tables S2-S4). Our RNAseq analysis also revealed that the lip1 mutation had a relatively strong effect on the gene expression under control and chilling stress conditions ( Figure 7A and Supplemental Tables S5-S7). Compared to the mpl1 and lip1 single mutations, the mpl1lip1 double mutations had a moderate effect on gene expression under control and chilling stress conditions ( Figure 7A and Supplemental Tables S8-S10). The RNA-seq analysis appears to be reliable because reductions in the expression of MPL1 and LIP1 were detected in the relevant mutant background (Supplemental Tables S3-S10). We subsequently validated the RNA-seq results by qRT-PCR analysis with three randomly selected genes ( Figure 7B  The differentially expressed genes in the mpl1, lip1, and mpl1lip1 plants encode proteins with diverse functions. In the mpl1 plants, 6 h of cold stress led to altered expression of nine genes and these genes encode proteins for cellular metabolism, protein synthesis and turnover, and responses to abiotic stresses (Supplemental Table S3. Exposure to cold stress for 48 h in the mpl1 plants resulted in altered expression of six genes, and these genes encode proteins for protein turnover, cellular metabolism, and plant development (Supplemental Table S4). It was apparent that short-term cold treatment (6 h) causes changed in the expression of more genes than the long-term cold treatment did (48 h) (Supplemental Tables S2 and S3). Cold treatment for 6 h altered the expression of 476 genes in the lip1 plants; a large portion of them encode proteins for cellular metabolism including lipid metabolism, and some of the genes encode components of the spliceosome for RNA processing, transcription factors for gene regulation, and enzymes for ROS detoxification (Supplemental Table S6). Cold treatment for 48 h in the lip1 plants had less effect on gene expression, and 169 genes displayed altered expression levels and these genes encode proteins with similar functions to those encoded by differentially expressed genes 6 h after cold treatment (Supplemental Table S7). Eighty-seven genes were differentially expressed in the mpl1lip1 plants after 6 h exposure to cold stress, and the expression of 73 genes was altered in the mpl1lip1 plants by cold treatment for 48 h (Supplemental Tables S9 and S10). The differentially expressed genes in the mpl1lip1 plants encode proteins involved in cellular metabolism, gene expression regulation, protein turnover, ROS detoxification and stress responses.

Discussion
Lipid biosynthesis and catabolism are critical for plant growth and development processes such as seed germination, seedling establishment, and seed development [18]. Triacylglycerols are important for membrane lipid breakdown, fatty acid b-oxidation, and plant survival under prolonged darkness conditions [19]. Triacylglycerol lipases carry out the catabolic reactions of triacylglycerol breakdown. It was shown that triacylglycerol lipases are important for seed germination, early seedling establishment, and normal growth and development of mature plants; however, their role in cold stress responses is not clear. In this study, we characterized two triacylglycerol lipases, MPL1 and LIP1, and defined their function in cold stress. The expression of both MPL1 and LIP1 was downregulated by cold stress, suggesting that they may function as negative regulators for cold stress. We suspect that there might be high level of functional redundancy between MPL1 and LIP1 for cold stress. To overcome this potential functional redundancy, we created the double mutant of mpl1lip1. To our surprise, the single-mutant plants of both mpl1 and lip1 displayed detectable phenotypic changes under cold stress. Both the mpl1 and lip1 plants were tolerant to chilling stress at multiple developmental stages. The mpl1lip1 plants were moderately more tolerant to the chilling stress conditions than the individual single-mutant plants, indicating that there is some level of additive effect of the mpl1 and lip1 mutations. Therefore, MPL1 and LIP1 display partial functional redundancy under chilling stress. We observed that the mpl1, lip1, and mpl1lip1 plants did not have altered responses to freezing stress and heat stress, suggesting that MPL1 and LIP1 have a specific function in chilling stress conditions. This observation is also consistent with their unique expression patterns under abiotic stresses. Because the mpl1lip1 double mutant does not show any detectable growth defects under both normal and cold stress conditions, the MPL1 and LIP1 might be ideal loci for breeding chilling-tolerant crops.
Mutations of MPL1 and LIP1 led to altered expression of genes under normal and cold stress conditions. The functions of those genes with differential expression levels in the mpl1, lip1, and mpl1lip1 plants under normal growth conditions were not apparent because these mutant plants did not show detectable phenotypic changes under normal conditions. Under cold stress conditions, there were more differentially expressed genes in the mutant plants after 6 h of cold treatment than in the plants exposed to a 48 h cold stress, presumably representing the changes in the expression of the early response genes. The differentially expressed genes under cold stress encode proteins with diverse functions, including metabolic enzymes for lipids, carbohydrates, and proteins; regulators of gene expression such as transcription factors; and components of the spliceosome. Collectively, the altered expression of these genes contributes to the increased chilling stress tolerance observed in the mpl1, lip1, and mpl1lip1 plants.
It has been shown that accumulation of lipid intermediates such as DAGs, free fatty acids, and membrane phospholipids is associated with premature cell death in growing leaves and floral organs [20]. Therefore, TAG synthesis or disruption of TAG breakdown can protect against free fatty acid-induced cell death in plant vegetative tissues. This, probably, at least in part, explains why the mpl1, lip1, and mpl1lip1 plants are tolerant to chilling stress. Furthermore, abiotic stress conditions including cold stress can cause altered membrane lipid composition through lipid remodeling to enhance the maintenance of membrane fluidity, stability, and integrity [21,22]. For example, heat stress induces the degradation of monogalactosyldiacylglycerol (MGDG) and chlorophyll in chloroplasts, leading to the accumulation of toxic lipid intermediates including DAGs, free fatty acids, and phytyl, which may damage plant tissues [23]. Under chilling stress conditions, the disruptions in TAG breakdown in the mpl1, lip1, and mpl1lip1 plants would have a profound impact on lipid metabolism including accumulation of TAGs and altered biological membrane lipid compositions. The accumulated TAGs could sequester the cold-induced toxic lipid intermediates to avoid cell death, thereby achieving improved chilling stress tolerance.

Chilling and Freezing Tolerance Assays
For chilling tolerance assay to examine the ability of seed germination and seedling development under light, seeds of the relevant genotypes were sown side by side on MS agar plates containing 0.6% agar. These agar plates were kept horizontally at 4 or 22 • C in growth chambers with a 16-h-light/8-h-dark photoperiod for the desired time.
For chilling tolerance assay to examine hypocotyl elongation in the dark, seeds of the relevant genotypes were sown side by side on MS agar plates containing 1.2% agar. These agar plates were then wrapped with aluminum foil and kept vertically at 4 or 22 • C in growth chambers for the desired time. Hypocotyl elongation at 4 • C relative to that at 22 • C was used as an indicator of sensitivity to chilling stress.
To evaluate freezing tolerance using electrolyte leakage assays, 3-week-old Col-0, mpl1, lip1, and mpl1lip1 plants were grown in soil at room temperature or at 4 • C under a 16-h-light/8-h-dark photoperiod for one week. Fully developed rosette leaves were used for electrolyte leakage measurements as described in [24][25][26].

Determination of Chlorophyll Content
Chlorophyll was determined as described in [27] with minor modifications. Seedlings were frozen in liquid nitrogen and ground to fine powder. Chlorophyll was extracted by incubating ground tissues in 80% acetone overnight at 4 • C in darkness and with continuous shaking. The contents of chlorophyll a and b were calculated as 7.49 A 664.9 + 20.3 A 648.2 .

RNA-seq Analysis and qRT-PCR Analysis
Fourteen-day-old Col-0, mpl1, lip1, and mpl1lip1 seedlings grown on MS medium (1× MS salts, 2% sucrose, 0.6% agar, pH 5.7) were treated at 4 • C for 0, 6, or 48 h and were used for total RNA extraction. Total RNA was isolated with the Universal Plant Total RNA Extraction Kit (BioTeke, Beijing, China) and treated with a TURBO DNA-free™ Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove any genomic DNA contaminants. mRNA-seq libraries were constructed following the standard Illumina protocols. There were three biological replicates per genotype. Illumina sequencing was performed in the Shanghai Center for Plant Stress Biology with an Illumina HiSeq 2500 system.
According to RNA-seq mapped reads and the reference annotated transcripts, transcriptomes were reconstructed for each sample using Cufflinks v2.2.1 [31]. Given the variable efficiency of mRNA enrichments and rRNA depletion kits in samples, these transcripts were masked in transcriptome constructions to improve the overall robustness of transcript abundance estimates. To obtain a high confidence of transcriptomes, the novel constructed transcript was filtered out when the abundance was lower than 20% (default is 10%) of the most abundant isoform for each gene. Then, all the constructed transcriptomes were merged with the reference annotated transcripts using Cuffmerge [31] to yield comprehensive re-annotated gene transcripts including known and novel annotated transcripts in our RNA-seq samples. Subsequently, significantly differentially expressed genes were predicted using Cuffdiff [31] between the controls and tested samples, using a twofold-change and multiple test p-value < 0.05.
For qRT-PCR analysis, 5 µg of total RNA was used to synthesize the first-strand cDNA with the Maxima First-Strand cDNA synthesis kit ( Thermo Fisher Scientific, Waltham, MA, USA) as described [32]. Each experiment had four biological replicates (two technical replicates for each biological replicate), and each experiment was repeated at least three times. The comparative cycle threshold method was applied, and UBQ5 was used as a reference gene. The gene-specific primers are listed in the Supplemental Table S11.

Phylogenetic Tree Construction
Close homologs of MPL1 and LIP1 from other plant species were identified through NCBI BLASTP searches. We also selected one lipase from the human genome. The full-length protein sequences of the MPL1, LIP1, and their homologs were aligned in MEGA11 with MUSCLE, and the phylogenetic tree was constructed with the neighborjoining method (Jones-Taylor-Thornton model; 1000 bootstrap replicates) [33]. The identities of the proteins in the phylogenetic tree are as follows: LIP1-X2 Brassica napus (accession number XP_013705879.