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Phospholipase-mediated phosphate recycling during plant leaf senescence

Genome Biology volume 25, Article number: 199 (2024) Cite this article

Abstract

Background

Phosphorus is a macronutrient necessary for plant growth and development and its availability and efficient use affect crop yields. Leaves are the largest tissue that uses phosphorus in plants, and membrane phospholipids are the main source of cellular phosphorus usage.

Results

Here we identify a key process for plant cellular phosphorus recycling mediated by membrane phospholipid hydrolysis during leaf senescence. Our results indicate that over 90% of lipid phosphorus, accounting for more than one-third of total cellular phosphorus, is recycled from senescent leaves before falling off the plants. Nonspecific phospholipase C4 (NPC4) and phospholipase Dζ2 (PLDζ2) are highly induced during leaf senescence, and knockouts of PLDζ2 and NPC4 decrease the loss of membrane phospholipids and delay leaf senescence. Conversely, overexpression of PLDζ2 and NPC4 accelerates the loss of phospholipids and leaf senescence, promoting phosphorus remobilization from senescent leaves to young tissues and plant growth. We also show that this phosphorus recycling process in senescent leaves mediated by membrane phospholipid hydrolysis is conserved in plants.

Conclusions

These results indicate that PLDζ2- and NPC4-mediated membrane phospholipid hydrolysis promotes phosphorus remobilization from senescent leaves to growing tissues and that the phospholipid hydrolysis-mediated phosphorus recycling improves phosphorus use efficiency in plants.

In brief

Work hard, plants try to absorb more phosphate from the soil. Work smart, plants effectively recycle phosphate from phospholipids from aging leaves.

Background

Phosphorus (P) is an essential macronutrient which is key for plant growth and crop production [1,2,3]. P is essential to all major metabolic processes in plants, such as photosynthesis and membrane biogenesis. But plant-available P, orthophosphate (Pi), is often limited in the soil [3,4,5]. Approximately 70% of global cultivated land is deficient in available P [3, 6]. To alleviate the P shortage, P fertilizers are widely used to increase crop yield, but the excessive use of P fertilizers increases production costs, leading to environmental pollution and accelerating depletion of finite P reserves [3, 7,8,9]. It becomes increasingly important that sustainable approaches are necessary to decrease the dependence on Pi fertilizers [3, 8, 10, 11]. This could be accomplished through enhancing the efficiency of P recycling within the plant to improve P use efficiency (PUE) and/or the plant’s P-acquisition efficiency from the soil [3, 12]. Understanding how plants recycle P will facilitate the development of strategies to design crops with increased PUE and decreased P dependency [11, 13, 14].

Leaves constitute a massive plant-specific tissue and play an important role in nutrient recycling [15,16,17]. In the final stage of leaf development senescence, nutrients, such as P, from old leaves are remobilized to growing tissues, such as young leaves and developing seeds [15, 16, 18, 19]. During senescence, chloroplasts are degraded, lipids are hydrolyzed by lipases to release acyl side chains, and carbon sources in lipids are recovered for acetyl-CoA metabolism [15, 20,21,22]. At the final stages of senescence, DNA and the plasma and vacuolar membranes are disintegrated. Comparative transcriptomic analyses showed that a number of genes, such as transcriptional factors, nucleases, lipases, phosphatases, and Pi transporters are upregulated during senescence, indicating an active regulation of P remobilization [15, 19, 23]. However, the molecular mechanism by which Pi is recycled from senescent leaves remains unclear [19, 24]. In particular, what are the key molecular players mediating P recycling from senescent leaves and their roles in plant growth and production?

Membrane phospholipids contain a significant pool of cellular P, constituting about one-third of the total P in plant cell [25, 26]. In response to P deficiency, phospholipids are decreased to release P to support plant growth [6, 26]. Two phospholipases, nonspecific phospholipase C4 (NPC4) and phospholipase Dζ2 (PLDζ2) are involved in the phospholipid hydrolysis as their expression is greatly induced by P deficiency [27,28,29,30]. PLDζ2 hydrolyzes the common phosphoglycerolipids, such as phosphatidylcholine (PC) [27, 28], whereas NPC4 hydrolyzes the most abundant phosphosphingolipids glycosyl inositol phosphate ceramides (GIPC) [30, 31]. The loss of phospholipids is partially compensated for by an increase of non-phospholipids, such as digalactosyldiacylglycerol (DGDG) and glucosylceramide (GlcCer) [32, 33]. The membrane lipid remodeling process to remobilize P from membrane phospholipids for other cellular uses is important for plant adaptation to P-deficient growth conditions [4, 26, 34]. How membrane phospholipids hydrolysis contributes to P recycling during leaf senescence has not been quantitatively characterized in plants and the key lipases involved in this process remains unknown. Here we report that NPC4 and PLDζ2 are the key enzymes that mediate P reutilization from senescent leaves, and they hydrolyze membrane phospholipids, promote leaf senescence, and enhance plant growth.

Results

Phospholipids and cellular P are decreased during leaf development and senescence

Rapeseed (Brassica napus) is a single-leaf alternate plant, and each leaf from the bottom to top is at a different developmental stage (Fig. 1a). We assayed the chlorophyll and P levels in each leaf of individual 160-day-old rapeseed plants at the bolting stage. The chlorophyll content is generally decreased from top to bottom leaves (Fig. 1b), which is a prominent feature of chloroplast damage during leaf senescence [15, 22]. At the same time, we found that the P level also decreased significantly during leaf senescence, including total P (TP), inorganic P (Pi), and lipid P (LP), especially at the late stage of senescence (Fig. 1c). The TP decrease is consistent with the notion that leaf nutrients are recycled and transferred to new tissues during senescence [15, 35], and the large reduction of LP suggests that membrane phospholipids are degraded for P recycling during leaf senescence.

Fig. 1
figure 1

Phospholipases activity increases to degrade membrane phospholipids during leaf senescence. a Phenotypes of 60-day-old rapeseed grown in a pot. b The chlorophyll contents in different stages leaves. Chlorophyll is extracted by 95% ethanol from 22 different stages leaves and quantitatively analyzed by microplate reader. Values are means ± SD (n = 3). c The levels of total phosphorus (TP), inorganic phosphorus (Pi) and lipids phosphorus (LP) in different stages leaves. Phosphorus level is determined in 22 different stages leaves. Values are means ± SD (n = 3). d The levels of Pod DGDG, SQDG, PC, GIPC, PA, and hCer during leaf senescence. Total lipids are extracted by solvent H (isopropanol/heptane/water 55:20:25, v/v/v) from leaves at 11 different stages of development and quantified by mass spectrometry. Values are means ± SD (n = 4). e Mfuzz clustering shows the changes in lipids levels that are categorized into eight clusters (C1–C8). Heatmap shows the relative lipids levels distribution in log2 for each cluster. C cluster. Values are means ± SD (n = 4). f MapMan indicates different types of lipid classes enriched in differential clusters (C1–C8). g Correlations among different lipid classes during leaf senescence. *Significant at P < 0.05; **significant at P < 0.01; and ***significant at P < 0.001 compared with the control based on the correlation analysis. Significant correlations were visualized using the “corrplot” function from the corrplot package in R. h The levels of glycerolipids and sphingolipids during leaf senescence. Total lipid extraction from natural senescent and dark-induced senescent leaves. Values are means ± SD (n =). i, j Phospholipase D (i) and nonspecific phospholipase C (j) activity assay during natural and dark-induced senescent leaves. Using PC as a substrate, detected by mass spectrometry, the PA level generated in the product represents the PLD activity (i) and the DAG level represents the NPC activity (j). Values are means ± SD (n = 3). The tick marks on the x-axis in b–e represent the leaves of different leaf ages from top to bottom of the plant. Different letters indicate differences at P < 0.05 using one-way ANOVA. GL glycerolipids, SL sphingolipids, PC phosphatidylcholine, PE phosphatidylethanolamine, PG phosphatidylglycerol, PI phosphatidylinositol, PS phosphatidylserine, PA phosphatidic acid, DAG diacylglycerols, MGDG monogalactosyldiacylglycerol, DGDG digalactosyldiacylglycerol, SQDG sulfoquinovosyldiacylglycerol, TAG triacylglycerols, GlcCer glucosylceramides, GIPC glycosylinositolphosphoceramides, hCer hydroxyceramide

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To determine lipid changes during leaf development and senescence, we analyzed the lipidomes of 11 leaves at different developmental stages. The lipidome included 460 different species of lipids in 15 classes of glycerolipids and 5 classes of sphingolipids (Fig. 1d and e; Additional file 1: Fig. S1a and Additional file 2: Table S1). Most of the lipids are increased and then decreased during leaf development (Fig. 1d and 1e; Additional file 1: Fig. S1a). The level of chloroplast membrane lipids (MGDG, monogalactosyldiacylglycerol; DGDG; PG, phosphatidylglycerol and SQDG, sulfoquinovosyldiacylglycerol) decreased earliest and most severely during leaf senescence (Fig. 1d; Additional file 1: Fig. S1a), with a reduction of 97%, 86%, 97%, and 66%. This corresponds to the disintegration of chloroplasts and the massive reduction of chlorophyll during leaf senescence (Fig. 1b). Meanwhile, phospholipids are greatly reduced in the later stages of senescence, such as a 50% or more reduction in glycerophospholipids (PC; PE, phosphatidylethanolamine and PS, phosphatidylserine) and 54% reduction in the phosphosphingolipids GIPCs that are enriched primarily in the plasma membrane (PM) (Fig. 1d; Additional file 1: Fig. S1a). On the other hand, the level of some lipid metabolic intermediates increased significantly in the late stages of senescent leaves, such as ninefold increase in phosphatidic acid (PA), twofold increase in diacylglycerols (DAG), and 2.5-fold increase in hydroxyceramide (hCer) (Fig. 1d; Additional file 1: Fig. S1a). These results indicate that the reduction of membrane phospholipids is the main source for the LP reduction during senescence.

Phospholipase activity increases during leaf senescence

To further analyze the lipid changes, we used Mfuzz clustering tool to divide 460 lipid species into 8 clusters, named lipid content clusters 1–8 (C1–C8), based on the pattern of changes during leaf development (Fig. 1e; Additional file 1: Fig. S1b and S1c; Additional file 2: Table S1). Meanwhile, cluster analysis is performed on lipid species in different clusters. The chloroplast lipids are mainly accumulated in C5 (the first type to decrease during senescence) (Fig. 1f) and phospholipids are mainly accumulated in C3 and C5 (Fig. 1f). Lipids including PA, DAG, and hCer are mainly accumulated in C8 (Fig. 1f). To determine the relationship between the levels of different lipid species during leaf senescence, we examined the correlations among lipid species during leaf senescence. The level of lipid metabolic intermediates PA, DAG, and hCer is negatively correlated with the level of chloroplast lipids and major membrane phospholipids (Fig. 1g). In addition, we compared the lipid changes between natural senescent and dark-induced senescent leaves (Fig. 1h; Additional file 1: Fig. S2a). The lipid changes in dark-induced senescent leaves are similar to those of natural senescent leaves. Membrane phospholipids and chloroplast lipids are degraded in large quantities while PA, DAG, and hCer levels increase significantly (Fig. 1h).

PLDs and NPCs hydrolyze membrane phospholipids to produce PA and DAG, respectively. In addition, NPC4 hydrolyzes GIPC to produce hCer [30]. Thus, the opposite changes in membrane phospholipids and intermediary lipids suggest an increase in phospholipase activity during leaf senescence. To test the phospholipase activity during senescence, we used soybean PC as substrate to assay PLD and NPC activities using proteins extracted from leaves at different stages (Fig. 1a, Additional file 1: Fig. S2a). Compared to young leaves, the total phospholipase activity is increased by 2.4-fold, substrate-PC levels decreased more in the experimental groups added with natural senescent and dark-induced senescent leaf protein extracts (Fig. 1i and j; Additional file 1: Fig. S2b). Among them, the activity of PLD (Fig. 1i) and NPC (Fig. 1j) is increased by 2.3- and 1.4–twofold, respectively, in senescent leaves. These results support that phospholipase activity increases during leaf senescence.

Correlations between transcriptomic and lipidomic changes during leaf senescence

To probe the biological processes and genes involved in mediating P recycling during leaf senescence, we performed transcriptome analysis of 22 leaves at different developmental stages of rapeseed. We used t-distributed stochastic neighbor embedding (t-SNE) analysis that grouped the transcriptomic data into three main clusters: Y, young stage; M, mature stage; and S, senescent stage (Fig. 2a). Mfuzz clustering divided the differentially expressed genes (DEGs) into eight clusters, named gene expression clusters C1–C8 (Fig. 2b–d; Additional file 1: Fig. S3 and Additional file 2: Table S2). Among them, the expression of the genes in C1 gradually increases with aging and leaf aging marker genes are enriched (Fig. 2e; Additional file 1: Fig. S3b). Expression of the genes in C5 is significantly induced in the late stages of leaves and autophagy marker genes are enriched (Fig. 2e). On the contrary, the expression of C7 genes is higher in young leaves, and some chloroplast matrix genes are enriched (Fig. 2e; Additional file 1: Fig. S3g).

Fig. 2
figure 2

Lipid metabolism-related genes play key role in regulating leaf senescence. a t-distributed stochastic neighbor embedding (t-SNE) analysis of the leaf transcriptome during senescence. Each different colored dot represents a different period of leaves. Based on t-SNE, three major sample clusters are identified: Y young stage, M mature stage, S senescence stage. b Mfuzz clustering shows the expression changes of DEGs that are categorized into eight clusters (C1–C8). Line charts show the expression pattern of each cluster during leaf senescence. Values are means. C cluster. c The co-expression network of genes in C1–C8. Each dot represents a gene. Each color represents a cluster. d Mfuzz clustering of the expression profiles of 43,699 differentially expressed genes (DEGs). Y young stage, M mature stage, S senescence stage. Each column is a different period of leaves. Each row is a different cluster. e Gene Ontology (GO) enrichment analysis of different clusters (C1–C8). Each column is a different cluster. Each row is a kind of GO term. White box indicates non-significant enrichment. The color of the box represents the enrichment level. f Correlation of gene expression clusters with lipid content clusters during leaf senescence. The horizontal axis is the gene expression cluster and the vertical axis is the lipid content cluster

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To identify candidate genes involved in membrane phospholipid hydrolysis in senescent leaves, we used MapMan functional categories to group genes enriched in differential co-expression modules (C1–C8) (Fig. 2e; Additional file 1: Fig. S3 and Additional file 2: Table S3). The results show that many genes enriched in C5 are related to cellular senescence-related pathways, such as leaf senescence, response to abscisic acid, and autophagy. In addition, some genes involved in the lipid metabolic process and cellular response to Pi starvation, such as NPC4 and PLDζ2 (Additional file 1: Fig. S4 and Additional file 2: Table S4), are also significantly enriched in C5 (Additional file 1: Fig. S4 and Additional file 2: Table S2).

To verify whether genes in C5 are related to membrane phospholipid metabolism during senescence, we performed the correlation analysis of gene expression clusters and lipid content clusters during leaf senescence. The gene expression of cluster C5 has a high positive correlation with the lipid content cluster C8, with a correlation coefficient of 0.97, whereas the lipid content is negatively correlated with clusters C3 and C5, with correlation coefficients of − 0.58 and − 0.74, respectively (Fig. 2f). Those results indicate that genes related to lipid metabolism in gene expression cluster C5 play a key role in membrane phospholipid metabolism during leaf senescence and that genes related to cellular response to Pi starvation play a role in P recycling from membrane phospholipids.

NPC4 and PLDζ2 are drastically induced during leaf senescence

To identify key genes mediating membrane phospholipid hydrolysis during leaf senescence, we performed gene ontology (GO) enrichment analysis of genes in gene expression cluster C5 (Fig. 3a). The results show that phosphorus deficiency stress-induced membrane phospholipid remodeling-related genes are significantly induced during leaf senescence (Additional file 1: Fig. S4). In addition, we analyzed the transcript level of genes in phospholipase families during leaf senescence in rapeseed (Additional file 1: Fig. S4 and S5; Additional file 2: Table S4). The transcript level of only NPC4 in the PLC family greatly increases at the late stages of senescence (Fig. 3b; Additional file 1: Fig. S4, S5 and Additional file 2: Table S4). PLDζ2 in the PLD family is markedly induced at late stages of leaf senescence whereas PLDβ2, PLDγ, and PLDζ1 are induced to varying degrees (Fig. 3b; Additional file 1: Fig. S4, S5 and Additional file 2: Table S4). To determine whether the expression level of NPC4 and PLDζ2 is associated with leaf senescence, we performed a correlation analysis of the expression of NPC4 and PLDζ2 with genes in cluster C5. The results show that NPC4 and PLDζ2 are highly correlated with marker genes in pathways related to leaf senescence, aging, and response to abscisic acid (Fig. 3c). In addition, the transcript level of NPC4 and PLDζ2 is highly correlated with that of marker genes in pathways related to Pi metabolism, such as phosphate ion homeostasis, acid phosphatases, and cellular response to Pi starvation during leaf senescence (Fig. 3c).

Fig. 3
figure 3

NPC4 and PLDζ2 are highly expressed in senescent tissues. a GO enrichment analysis of cluster 5 in senescent stage. The count number represents the number of genes enriched in a GO term and the color represents the range of p-value. The genes of cluster 5 are involved in metabolic processes, including lipid metabolic process, cellular response to phosphate starvation, chlorophyll catabolic process etc. b Expression of NPC4 and PLDζ2 during leaf senescence of rapeseed. Gene expression data are from BnTIR (http://yanglab.hzau.edu.cn/). c Co-expression analysis of NPC4, PLDζ2 and leaf senescence marker genes in cluster 5. Each circle represents a gene. Different color represents different GO term. d, e Expression level of NPC4 and PLDζ2 during leaf senescence by quantitative real-time PCR. Total RNA was extracted from leaves of different periods and locations. Values are means ± SD (n = 3). Y young leaves, M mature leaves, ES early-stage senescing leaves, LS late-stage senescing leaves. B basal section, M middle section, T tip section. f Expression level of lipid metabolism genes in dark-induced senescent leaves by quantitative real-time PCR. Total RNA was extracted from dark-induced senescent leaves for 5 days. Values are means ± SD (n = 3). g Expression of NPC4 and PLDζ2 in different tissues at different stages of rapeseed. Gene expression data are from BnTIR (http://yanglab.hzau.edu.cn/). The dots in the yellow circle are the leaves and silique wall in the last stage of development. h Correlation of NPC4 and PLDζ2 expression levels with lipid levels during leaf senescence. The hexagon represents a gene. Each circle represents a lipid. The gray line represents the negative correlation, the orange line represents the opposite, and the thickness of the line represents the strength of the correlation

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We further verified that NPC4 and PLDζ2 are highly expressed in senescent leaves by qPCR (Fig. 3d, e). In addition, other membrane lipid remodeling-related genes in response to phosphorus deficiency are induced in senescent leaves (Additional file 1: Fig. S4, S5 and Additional file 2: Table S4), such as phosphatase genes (phosphatidic acid phosphohydrolase, PAH; phosphate starvation-induced gene 2, PS2; glycerophosphodiester phosphodiesterase, GDPD; and phosphoethanolamine/phosphocholine phosphatase 1, PEPC1), glycolipid synthesis genes (glucosylceramide synthase, GCS; monogalactosyl diacylglycerol synthase, MGD; digalactosyldiacylglycerol synthase, DGD; and sulfoquinovosyltransferase, SQD), and TAG synthesis gene (diacylglycerol acyltransferase, DGAT) [26, 32]. Consistently, the expression of those lipid genes in metabolism also increases in dark-induced senescent leaves (Fig. 3f). To determine the expression specificity of NPC4 and PLDζ2, we analyzed the transcriptome data in 13 tissues at 91 different periods of rapeseed and found that NPC4 and PLDζ2 are mainly expressed at high levels in senescent tissues, such as senescent leaves and senescent silique wall (Fig. 3g), and their expression is associated with other membrane phospholipid remodeling genes, such as PAH, GCS, MGD, DGD, and SQD, that are also highly expressed in aging tissues (Additional file 1: Fig. S6a and S6b).

In addition, we analyzed the correlation between the expression of these two genes, phospholipases highly induced during leaf senescence, and lipid levels. The expression level of NPC4 and PLDζ2 is negatively correlated with that of the membrane phospholipids (PC, PE, PS, and GIPC) and chloroplast lipids (MGDG, DGDG, and PG) but positively correlated with that of PA, DAG, and hCer (Fig. 3h; Additional file 1: Fig. S6c and Additional file 2: Table S5). These results indicate that the high expression of NPC4 and PLDζ2 are closely related to the reduction of membrane phospholipid levels during leaf senescence, and the genes related to membrane lipid remodeling during P deficiency play a key role in the recycling of LP during leaf senescence.

NPC4 and PLDζ2 promote leaf senescence

To test the function of NPC4 and PLDζ2 during leaf senescence and P recycling, we knocked out NPC4 and PLDζ2 by CRISPR/Cas9 in rapeseed (Additional file 1: Fig. S7a and S7b). There are two copies of NPC4 in the rapeseed genome that are highly similar in sequences. To edit both NPC4s, we used CRISPR-P2.0 to target the second exon of the homology segment (Additional file 1: Fig. S7a). PLDζ2 is edited at the fourth exon (Additional file 1: Fig. S7b). Five lines of NPC4 homozygous mutants and four lines of PLDζ2 homozygous mutants were obtained. These mutations result in frameshifts and premature termination during translation. In addition, we produced transgenic rapeseed lines overexpressing NPC4 and PLDζ2 driven by the 35S promoter and obtained three lines of NPC4-overexpression (OE) and four lines of PLDζ2-OE (Additional file 1: Fig. S7c). Furthermore, we generated three independent lines of NPC4/PLDζ2-OE by crossing NPC4-OE and PLDζ2-OE (Additional file 1: Fig. S7c) and NPC4/PLDζ2-KO mutants by crossing NPC4-KO and PLDζ2-KO (Additional file 1: Fig. S7d).

Phenotypic analysis of plants grown for 6 weeks under sufficient P conditions shows that compared with wild type (WT), OE of NPC4 and PLDζ2 single or double gene promotes leaf senescence as indicated by increased leaf yellowing (Fig. 4a, Additional file 1: Fig. S8a, S9 and S10). On the contrary, the loss of a single NPC4 or PLDζ2 and NPC4/PLDζ2-KO double mutant delays leaf senescence. In addition, we observed that NPC4-KO and PLDζ2-KO delay leaf senescence in dark-induced leaf senescence (Additional file 1: Fig. S8a). The increased leaf senescence in the NPC4-OE and PLDζ2-OE plants is indicated by the significantly decreased chlorophyll (Fig. 4b; Additional file 1: Fig. S9a and S10a) and protein contents (Fig. 4c; Additional file 1: Fig. S9b and S10b), compared with WT. In addition, we monitored the changes in the level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) located in the chloroplast, and the results support the opposite effects of the KO and OE of NPC4 and PLDζ2 on leaf senescence (Fig. 4d; Additional file 1: Fig. S9c and S10c).

Fig. 4
figure 4

Membrane lipid remodeling genes play an important role in promoting leaf senescence. a Plant growth under normal condition for 6 weeks. Senescent phenotype arises from second true leaf. b The chlorophyll content during leaf senescence. Values are means ± SD (n = 4). The chlorophyll was extracted from second true leaf. c The protein content during leaf senescence. Values are means ± SD (n = 3). The protein was extracted from second true leaf. d Coomassie brilliant blue staining of rubisco large subunit (Rbc L) during leaf senescence. The protein was extracted arises from second true leaf. e Volcano plots show significant changes in gene expression in OE42 compared with WT. Total RNA was extracted from the second leaf at the five-leaf-stage. Genes marked with lines are genes associated with leaf senescence. f GO enrichment analysis of upregulated genes in OE42 compared with WT. g Expression level of aging- and photosynthesis-related genes by quantitative real-time PCR. Total RNA was extracted from the second leaf at the five-leaf-stage. Values are means ± SD (n = 3). h The leaf ABA content. ABA was extracted from the second leaf at the 5-leaf-stage. Values are means ± SD (n = 4). Different letters indicate differences at P < 0.05 using one-way ANOVA. *Significant at P < 0.05; **Significant at P < compared with WT under the same condition, based on Student’s t-test

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To further explore how NPC4 and PLDζ2 promote leaf senescence, we analyzed the effect of NPC4 and/or PLDζ2 alterations on the transcriptome using the second true leaf of five-leaf stage plants. The results show that the KO and OE of NPC4 and PLDζ2 result in changes in the expression of many senescence-responsive genes (Fig. 4e; Additional file 1: Fig. S8b, S9d, S9e, S10d and S10e). Compared with WT, NPC4-OE plants display upregulated genes enriched in pathways involved in leaf senescence and response to abscisic acid (Additional file 1: Fig. S9f) whereas downregulated genes in NPC4-OE plants are enriched in pathways in photosynthesis, chlorophyll binding, and chlorophyll biosynthetic processes (Additional file 1: Fig. S9g). In contrast, NPC4-KO plants have downregulated genes enriched in the lipid metabolic process (Additional file 1: Fig. S9h). The gene expression changes are verified with qPCR and compared with WT, aging positive regulatory genes (SAG12, GSR1, RD29B, ORE1, ATG8F) are upregulated in NPC4-OE plants, while significantly downregulated in NPC4-KO, and photosynthesis-related genes (GPRI1, LHCB6, PSAF, DRN1, etc.) are downregulated in NPC4-OE and upregulated in NPC4-KO (Additional file 1: Fig. S9i).

Compared with NPC4, the changes in the expression of PLDζ2 had less effect on the expression changes of other genes, which may be due to partial functional redundancy of PLDζ1 that is also mildly induced by senescence (Fig. 3f; Additional file 1: Fig. S4 and S5c), and these two genes had some functional redundancy. However, compared with WT, some senescence-responsive genes, such as NOL, SAG21, and SAG101 are increased in PLDζ2-OE31, and chloroplast-related genes, including PGK3, SPS1, and PGR5 are significantly upregulated in PLDζ2-KO mutants (Additional file 1: Fig. S10f). The upregulated genes in NPC4/PLDζ2-OE42 are significantly enriched in processes such as leaf senescence, chlorophyll catabolic process, and response to abscisic acid (Fig. 4f). Significantly upregulated genes in NPC4/PLDζ2 double mutants include pathways of fatty acid synthesis and carbohydrate metabolism (Additional file 1: Fig. S8d). We further found that compared with WT, positive regulatory genes of aging (e.g., SAG20, VIN2, RD29B, NAP, SGR1) are significantly upregulated in OE42 but significantly downregulated in C89. Photosynthesis-related genes (e.g., CORI1, CHLM, PSAF, MNB) are significantly downregulated in NPC4-OE and significantly upregulated in NPC4-KO (Fig. 4g). ABA is a key hormone that regulates plant senescence [15, 36]. We detected the ABA level in leaves and the results show that the ABA level in NPC4/PLDζ2-OE is doubled compared with WT but is decreased by 46% in NPC4/PLDζ2-KO (Fig. 4h). These results indicate that NPC4 and PLDζ2 are involved in phospholipid hydrolysis during plant leaf senescence.

NPC4 and PLDζ2 enhance P recycling and plant growth

To probe the impact of NPC4 and PLDζ2 on lipid metabolism, we detected the lipid levels in rapeseed leaves. Compared with WT, the level of membrane phospholipids (PC, PE, PS, GIPC) in NPC4- and/or PLDζ2-OE leaves is decreased by about 5–10%, while they are increased by about 5–20% in NPC4 and/or PLDζ2-KO mutants (Fig. 5a; Additional file 1: Fig. S11a and S11b). Intermediate metabolic lipids (PA, DAG, hCer) are increased by about 5–15% in NPC4 and/or PLDζ2-OE leaves and are decreased by about 3–8% in NPC4- and/or PLDζ2-KO mutant (Fig. 5a; Additional file 1: Fig. S11a and S11b). Meanwhile, the level of LP is consistently higher in NPC4- and PLDζ2-KO leaves, but lower in OE leaves, which is consistent with the changes in lipidomes (Fig. 5b, Additional file 1: Fig. S11c and S11d). In addition, we analyzed the leaf transcriptome, followed by qPCR validation. Compared with WT, NPC4- and PLDζ2-OE plants have a higher expression level of lipid metabolism genes, but their KO plants have a lower expression level of these genes (DGD2, SQD2, TAG1), phosphorus metabolism genes (GDPD, PS2, PAP), and phosphorus homeostasis regulation genes (CAX3, PHT, PHO1) (Fig. 5c; Additional file 1: Fig. S11e-S11h). These results indicate that NPC4 and PLDζ2 promote membrane phospholipid hydrolysis to reduce the LP level to increase metabolic cellular P levels in plants for P reutilization.

Fig. 5
figure 5

Membrane lipid remodeling genes improve phosphorus use efficiency and promote plant growth. a The levels of lipids in leaves. Total lipids were extracted from the second leaf at the five-leaf-stage. Values are means ± SD (n = 4). b The levels of total phosphorus (TP), phosphorus inorganic (Pi), and lipids phosphorus (LP) in the different stages of leaves. The expanded leaf is numbered from bottom to top, with the first leaf being the oldest. Values are means ± SD (n = 3). c Expression level of lipid metabolism and phosphorus metabolism genes in plant by quantitative real-time PCR. Total RNA was extracted from the second leaf at the five-leaf-stage. Values are means ± SD (n = 3). d Phenotypes of plants grown in nutrient-rich nutrient soil for 3 weeks. The expanded leaf is numbered from bottom to top, with the first leaf being the oldest. e, f, g Plant height (e), number of true leaves (f), and dry weight (g) of 3-week-old plants. Values are means ± SD (n = 5). h The model of recycling and using phosphorus from aging leaves to new tissues. NPC4 and PLDζ2 are induced to express abundantly during plant senescence, hydrolyzing phospholipids to release phosphorus and neutral lipids (DAG and hCer). At the same time, the glycolipid synthase (GS) gene is also induced to further catalyze downstream lipid metabolism. The released phosphorus is transferred to young tissues by phosphorus transporters (PT) for reuse in cellular processes that require phosphate, such as synthesis of DNA, RNA, ATP, phospholipids, etc. Different letters indicate differences at P < 0.05 using one-way ANOVA. *Significant at P < 0.05; **significant at P < compared with WT under the same condition, based on Student’s t-test

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To monitor the effect of NPC4 and PLDζ2 on cellular P utilization, we measured the P levels in each leaf of rapeseed at the seven-leaf stage (Fig. 5b, Additional file 1: Fig. S11c and S11d). Compared with WT, TP and Pi in older leaves of NPC4- and PLDζ2-OE plants are decreased by about 4–8% and 6–11%, whereas TP and Pi are increased by about 6–13% and 5–25% in older leaves of NPC4- and PLDζ2-KO plants. In contrast, the upper young leaves of NPC4- and PLDζ2-OE plants are about 6–11% and 8–14% lower whereas those of NPC4- and PLDζ2-KO are about 4–7% and 5–8% lower in TP and Pi, respectively, than WT (Fig. 5b; Additional file 1: Fig. S11c and S11d). And we calculated the biomass produced per milligrams of TP, Pi, and LP in each leaf of rapeseed at the seven-leaf stage (Additional file 1: Fig. S11i-S11k), that is, the phosphorus utilization efficiency. Compared with WT, various PUEs in older leaves of OE lines were significantly improved, while those in the mutants were significantly decreased (Additional file 1: Fig. S11i-S11k). These results suggest that NPC4 and PLDζ2 promote of the remobilization of P from senescent leaves to young leaves, resulting in higher PUE in older leaves and lower PUE in young leaves (Additional file 1: Fig. S11k).

We further analyzed the effect of NPC4 and PLDζ2 alteration on plant growth (Fig. 5d; Additional file 1: Fig. S12). Compared with WT, OE of NPC4 and PLDζ2 increases plant height by 18–84% after 1 week of germination (Additional file 1: Fig. S12a and S12b), and the effects of these two genes are additive. At the same time, NPC4 and PLDζ2 promote the growth of plants at various stages under potted and hydroponic conditions (Fig. 5d; Additional file 1: Fig. S12c-S12e). Compared with WT, the plant height (Fig. 5e), number of true leaves (Fig. 5f), and dry weight (Fig. 5g) of NPC4/PLDζ2-overexpressed plants are increased by 17%, 17%, and 23%, while the NPC4/PLDζ2-mutants are decreased by 14%, 10%, and 26%. Those results further support that NPC4 and PLDζ2 mediate the removal of membrane LP from senescing leaves to promote Pi reuse in new tissues and plant growth (Fig. 5h).

NPC4/PLDζ2-mediated P recycling from senescing leaves is conserved in plants

To demonstrate the conservation of NPCs and PLDζs function in plants, we analyzed the evolution of NPCs and PLDζs of dicotyledonous species Arabidopsis, cotton, and soybean and monocotyledonous species wheat, rice, and maize; we found that both NPC4 and PLDζ2 genes are conserved in plants (Additional file 1: Fig. S13a and S13b). And analysis of the expression profiles of NPC4 and PLDζ2 in various tissues and stress conditions indicate that NPC4 and PLDζ2 are highly expressed in senescent leaves (yellow dots) in various plants (Additional file 1: Fig. S13c-S13g). Meanwhile, compared with green leaves, the expression levels of NPC4 and PLDζ2 in old leaves are significantly increased revealed by qPCR verification (Fig. 6a). To probe whether the phospholipase-mediated P recycling in leaf senescence is conserved in different plant species, we assessed the P level in leaves of plants at different developmental stages. The levels of TP, Pi, and LP in the senescent leaves of different plants are significantly lower than those in the young leaves (Fig. 5b). At the same time, the levels of TP, Pi, and LP are also decreased significantly during leaf senescence of deciduous trees, such as Populus tomentosa, Broussonetia papyrifera, Platanus orientalis, and Giokgo biloba (Additional file 1: Fig. 14a). Lipidomic analyses show that chloroplast lipids (PG, MGDG, DGDG) in the aging leaves of various plants are decreased by nearly 90% or more, and membrane phospholipids levels (PC, PE, PS, GIPC) are decreased by about 80–90%, whereas the level of PA, DAG, and hCer keeps a relatively high level all the time (Fig. 6c; Additional file 1: Fig. 14b). These results suggest that the phospholipase-mediated P recycling during leaf senescence is conserved in plants.

Fig. 6
figure 6

Alteration of NPC4 and PLDζ2 impacts phosphorus, lipid and gene expression levels during leaf senescence. a Expression of NPC4 and PLDζ2 during leaf senescence of different plants. Gene expression was quantified by quantitative real-time PCR. Values are means ± SD (n = 3). b The levels of total phosphorus (TP), inorganic phosphorus (Pi) and lipids phosphorus (LP) during leaf senescence in different plants. Values are means ± SD (n = 3). c The levels of glycerolipids and sphingolipids during leaf senescence in different plants. Values are means ± SD (n = 4)

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Discussion

In this study, we discovered a mechanism of P recycling during leaf senescence. We quantitatively show that over 90% of LP, accounting for more than one third of TP, is recycled from senescent leaves (Fig. 1d, 1h, 6b and 6c; Additional file 1: Fig. S1a and S14). Increased phospholipase activity is directly involved in the hydrolysis of phospholipids in this process during leaf senescence (Fig. 1i and 1j; Additional file 1: Fig. S2b). NPC4 and PLDζ2 hydrolyze membrane phospholipids to release LP from senescent leaves to promote P reuse and plant growth (Fig. 5a–g). The expression of NPC4 and PLDζ2 increases greatly in senescent leaves (Fig. 3b–e and 6a; Additional file 1: Fig. S13c-S13g) and the transcript level of NPC4 and PLDζ2 is negatively correlated with the level of membrane phospholipids during leaf development (Fig. 3h). The overexpression of NPC4 and PLDζ2 significantly reduces the level of phospholipids in leaves. In addition, the increased expression of NPC4 and PLDζ2 is associated with increased expression of P metabolism and transporter genes (Fig. 5c), suggesting that P released from phospholipids is actively transferred to young tissues. These results indicate that NPC4 and PLDζ2 promote the decrease in LP content, increase inorganic P level, accelerate P recovery from old leaves, and enhance plant growth.

Previous studies show that NPC4 and PLDζ2 are involved in membrane lipid remodeling in plant response to P deficiency [27,28,29,30, 37]. Under limited P conditions, the amount of membrane phospholipids is reduced and that of glycolipids is increased to partially compensate for the membrane function [26, 32]. During leaf senescence, our results show that membrane phospholipids are greatly decreased and the expression of some genes related to glycolipid synthesis is also increased (Fig. 1d; Additional file 1: Fig. S1a and S4). However, unlike the membrane lipid remodeling under P deficiency, we did not detect the accumulation of non-phospholipids, except for a small increase in TAG levels (Fig. 1d; Additional file 1: Fig. S1a and S4). This could be due to the recycling of carbon sources in lipids by the extensive destruction of cell membranes during leaf senescence (Additional file 1: Fig. S15 and S16). Previous studies have shown that lipid turnover during senescence is a carbon source recycling that induces lipase activity to hydrolyze acyl groups to form acetyl-CoA through fatty acid β-oxidation, thereby completing the recycling of carbon sources in lipids [18, 20, 21, 23, 38]. And a series of genes mediating carbon source recycling in membrane lipids are induced during leaf senescence (Additional file 1: Fig. S15). This is consistent with the undetectable accumulation of large amounts of non-phospholipids. But the degradation of marker lipid SQDG in response to phosphorus deficiency was significantly slower than that of MGDG and DGDG during leaf senescence (Additional file 1: Fig. S1), and the SQDG synthesis gene SQD was significantly induced during leaf senescence (Additional file 1: Fig. S4). These results suggest that membrane lipid remodeling occurs during leaf senescence.

The opposite effects displayed by NPC4/PLDζ2-KO and OE on rapeseed leaf senescence and plant growth show that NPC4- and PLDζ2-mediated P recycling from senescent leaves are key mediators in plant PUE, and NPC4 improves PUE and increases rapeseed yield [39, 40]. At the same time, NPC4/PLDζ2 hydrolyzes phospholipids to produce DAG, hCer, and PA, which can serve as second messengers to adjust plant growth and response to stresses, thereby promoting plant growth. In modern crop production, increasing amounts of P fertilizers are applied to achieve high yields, but this practice is unsustainable due to the finite P supply and environmental pollution [3, 4, 12]. Substantial progress has been made to increase P acquisition efficiency from the soil, but little is known about increasing PUE [9, 41, 42]. Given the limited, finite supply of P for crop production, improving PUE of plants becomes increasingly important for decreasing the dependence on Pi fertilizers and for sustainable crop production [3, 6]. Thus, it will be of great interest to explore how the positive PUE effectors NPC4 and PLDζ2 could be used to improve PUE to reduce the use of P fertilizers for crop production.

Conclusions

This study reports a mechanism of cellular phosphorus recycling mediated by membrane phospholipid hydrolysis during leaf senescence. We show that phospholipids and cellular P levels decrease about 90% during leaf senescence. Phospholipase activity is strongly increased in leaf senescence. The key phospholipase genes, NPC4 and PLDζ2, are highly induced during leaf senescence, hydrolyzing phospholipids, and the released phosphorus is remobilized to young tissues in large quantities during leaf senescence. We also show that the phosphorus recycling from senescent leaves mediated by phospholipid hydrolysis is conserved in plants (from Arabidopsis to crops and trees). We conclude that phospholipid hydrolysis mediated by PLDζ2 and NPC4 contributes to phosphorus recycling and improves PUE in plants. NPC4 and PLDζ2 could be used to improve PUE to reduce the use of P fertilizers for crop production.

Methods

Plant materials and growth conditions

The rapeseed cultivar Westar was used for all physiological experiments and plant transformations. Hydroponic experiments were conducted using a modified plant culture Hoagland solution containing 5 mM KNO3, 1 mM KH2PO4, 2 mM MgSO4, 5 mM Ca(NO3)2, 46 μM H3BO3, 0.32 μM CuSO4, 0.77 μM ZnSO4, 9.14 μM MnCl2, and 0.37 μM Na2MoO4 with 50 μM ethylenediaminetetraacetic acid (EDTA)-Fe (II). The pH of the hydroponic solution is 5.8 and the hydroponic solution is replaced once a week. Rapeseed plants were grown in a growth room with a 16-h light/8-h dark cycle at 25/21 °C, 50% humidity, and 200 µmol m−2 s−1 of light intensity. The transgenic materials of NPC4 came from our laboratory [39], and the transgenic material of PLDζ2 was derived from Agrobacterium-mediated transformation as for NPC4. In addition, we generated independent lines of NPC4/PLDζ2-OE by crossing NPC4-OE and PLDζ2-OE and NPC4/PLDζ2-KO mutants by crossing NPC4-KO and PLDζ2-KO. Primers used for the PCR verification of OE and mutant are listed in Additional file 2: Table S6. In field and pot experiments, transgenic seeds were sown in a random fashion with three replicates for WT, CRISPR-edited, and OE lines. Twenty plants were planted in each area, equally spaced from each other. Photographic phenotype analysis was performed when plants were potted at different time points. In pot experiments, plants were grown in nutrient-rich nutrient soil throughout the growth period.

Phosphorus extraction and content determination

Pi determinations and elemental analysis were performed as described previously [43, 44]; 10 mg of freeze-dried tissues were homogenized to 100 μL 1% (v/v) acetic acid. The extracts were centrifuged twice at 12,000 g for 15 min at 4 °C. Samples were diluted and a 90-μL aliquot was combined with 210 μL 0.35% (w/v) NH4MoO4, 1.4% (w/v) ascorbic acid in 1 N H2SO4, and incubated in the dark for 60 min at 37 °C. A standard curve was constructed using dilutions of KH2PO4. The absorbance of the reaction products was measured at 820 nm. Total P was extracted from 10 to 20 mg of freeze-dried tissue by digesting in H2SO4-H2O2 at 100 °C for 30 min. After the sample was completely clarified and cooled, an equal volume of deionized water was added. Samples were diluted as necessary before combining 150 μL with 50 μL malachite green and polyvinyl alcohol mixture and allowed to stand for 8 min. The absorbance of the reaction was measured at 650 nm and the total P concentrations of the samples were extrapolated from a standard curve constructed using standard solutions of KH2PO4. For the lipid phosphorus extraction assay [45], 50 mg sample was placed in 2-mL centrifuge tube A. Add 1 mL of chloroform:methanol:formic acid (12:6:1, v/v/v) mixture, vortex and mix well, and centrifuge at 1200 g for 10 min. Transfer the supernatant to a new 10-mL tube B. Then add 1.26 mL of chloroform:methanol:water (1:2:0.8, v/v/v) mixture to tube A, vortex and mix well, centrifuge at 1200 g for 10 min, and collect the supernatant in tube B. Add 1.9 mL of chloroform to tube B, vortex to mix well, and then centrifuge at 1100 g for 10 min. Discard the supernatant, dry the liquid in tube B with nitrogen, and lipid phosphorus content was determined according to the total phosphorus determination method.

Lipid extraction and lipid analysis by mass spectrometry

Lipids were extracted as described previously [46, 47]. Briefly, 10 to 30 mg of freeze-dried leaves at different stages of development were homogenized with 3 ml extraction solvent H (isopropanol/heptane/water 55:20:25). The supernatant was collected after repeated extraction until the leaves were colorless and dried under a stream of nitrogen gas. Samples were heated in 1 mL of tetrahydrofuran (THF)/methanol/water (2:1:2 v/v/v) containing 0.1% formic acid and dissolved by ultrasound sonication, then centrifuged at 500 g for 10 min to remove the insoluble substance. Total lipids were analyzed using an Exion UPLC system coupled with a triple quadrupole/ion trap mass spectrometer (6500 Plus QTRAP; SCIEX) according to a previous method with modifications [30, 47].

Phospholipase activity assays

The reaction mixture contained total protein from leaves of different developmental stages, PC (Avanti Polar Lipids Inc. 840054P) substrate and assay buffer (25 mM HEPES, pH 7.5, 10 mM CaCl2, and 10 mM MgCl2). PC was emulsified in a reaction buffer by sonication on ice for 5 min; 10 μg of total leaf protein was added to the reaction mixture in a final volume of 200 µL. The reaction was incubated at 30 °C for 30 min, and stopped by adding of 200 µL of chloroform followed by vigorous vortexing. The sample was centrifuged at 12,000 g and 200 µL of upper phase was used for mass spectrometry to detect the changes in various lipid levels [29, 30, 48].

RNA extraction and real-time PCR

Total RNA was extracted from leaves at different stages of development using Trizol. The RNA samples were sent to the sequencing company for RNA-seq library construction and sequencing [49]. After the sequencing is completed, we calculated the gene expression as TPM (transcripts per kilobase of exon model per million mapped reads) value. The corresponding Arabidopsis gene function annotation, gene name, and other information were obtained through homologous comparison. According to the TPM value of each gene, differential gene expression analysis, clustering of differentially expressed gene expression patterns, and GO enrichment analysis were performed, and the remaining RNA was quantitatively analyzed and verified for the target gene after reverse transcription. Isolated RNA was used as a template for cDNA synthesis through reverse transcription using an iScript kit (Bio-Rad). PCR products were quantitatively monitored by SYBR green fluorescent labeling of double-stranded DNA using CFX Connect (Bio-Rad). The expression level was normalized to that of ACTIN. PCR reactions were as follows: one cycle of 95 °C for 1 min, 45 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s, and final extension of DNA at 72 °C for 5 min. The real-time PCR primers for genes are listed in Additional file 2: Table S6.

Induction of leaf senescence

Rapeseed leaves were covered by tinfoil shading to induce darkness-induced senescence [50]. Non-senescent leaves were wrapped with tinfoil without damaging the leaves. The unshaded leaves at the same period were used as a control. Leaf senescence was determined when the leaves gradually turned yellow after several days of dark treatment. Harvested leaves were immediately frozen in liquid N2 and stored at − 80 °C for lipids levels and gene expression analysis.

Extraction and determination of chlorophyll and protein

Fresh or freeze-dried leaves were weighed and ground in liquid nitrogen using a mortar and pestle. The pulverized leaf tissue was added to 95% ethanol, vortexed, and incubated at room temperature in the dark for 30 min and repeatedly extracted until the leaves turned colorless. The solution was vortexed and centrifuged (3000 g, 10 min) and the absorbance of the supernatant was measured at 663 and 645 nm to determine the content of chlorophyll based on leaf weight [50].

For protein extraction, leaves at different stages of development were homogenized in a chilling buffer A (50 mM Tris–HCL, pH 8.0, 1 mM ethylenediaminetetraacetic acid, 10 mM KCl, 2 mM DTT, 0.5 mM PMSF, and 0.5 M sucrose). The homogenate was centrifuged at 6000 g for 10 min to obtain supernatant. The amount of purified protein was measured with a protein assay kit (Bio-Rad). Equal volumes of total proteins were separated by 10% w/v SDS-PAGE and then stained by Coomassie brilliant blue solution.

Statistical analysis

Values are means ± SD. Different lower letters indicate differences at P < 0.05 among genotypes during different grow conditions using one-way ANOVA. *Significant at P < 0.05; **significant at P < 0.01; and ***significant at P < 0.001 compared with the control based on Student’s t-test.

Availability of data and materials

Transcriptome data of leaves at different stages data were downloaded BnTIR (http://yanglab.hzau.edu.cn/) [49]. RNA sequencing data generated in this study are available at Gene Expression Omnibus (GEO) under accession numbers PRJNA1025340 [51].

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Peer review information

Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Review history

The review history is available as Additional file 3.

Funding

This work is supported by the National Natural Science Foundation of China (32372046), National Natural Science Foundation for Young Scholars of China (32300234), Fundamental Research Funds for the Central Universities (2662022ZKYJ003), Higher Education Discipline Innovation Project (B20051), and China Postdoctoral Science Foundation (2023M731230).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China

    Bao Yang, Zengdong Tan, Jiayu Yan, Zhewen Ouyang, Ruyi Fan, Yefei Lu, Yuting Zhang, Xuan Yao, Hu Zhao, Shaoping Lu & Liang Guo

  2. Department of Biology, University of Missouri, St. Louis, MO, 63121, USA

    Bao Yang, Ke Zhang & Xuemin Wang

  3. Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA

    Bao Yang, Ke Zhang & Xuemin Wang

  4. Yazhouwan National Laboratory, Sanya, 572025, Hainan, China

    Zengdong Tan, Yuting Zhang, Xuan Yao & Liang Guo

Authors
  1. Bao Yang
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  2. Zengdong Tan
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  3. Jiayu Yan
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  4. Ke Zhang
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  7. Yefei Lu
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  8. Yuting Zhang
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Contributions

L.G., B.Y., and S.L. designed and supervised the study. B.Y., Z.T., K.Z., J.Y., Y.O., R.F., Y.L., and H.Z. performed the experiments. B.Y. and Z.T. analyzed the data. B.Y. and X.W. wrote the manuscript. L.G., X.W., X.Y., and S.L. revised the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Shaoping Lu or Liang Guo.

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Supplementary Information

13059_2024_3348_MOESM1_ESM.pdf

Additional file 1: Fig. S1. Change of lipids levels during leaf senescence in rapeseed. Figure 2 Phospholipases activity are increased in senescent leaves. Fig. S3 Gene expression change patterns in C1-4 and C6-8. Fig. S4 Expression of lipid metabolism-related genes during leaf senescence of rapeseed. Fig. S5 Expression of membrane lipid remodeling-related genes is significantly increased during leaf senescence. Fig. S6 Expression of membrane lipid remodeling-related genes is significantly increased in senescent tissues. Fig. S7 Relative gene expression level and genotype analysis of rapeseed. Fig. S8 NPC4 and PLDζ2 play an important role in regulating leaf senescence. Fig. S9 NPC4 play an important role in regulating leaf senescence. Fig. S10 PLDζ2 play an important role in regulating leaf senescence. Fig. S11 NPC4 and PLDζ2 can improve phosphorus use efficiency in rapeseed. Fig. S12 NPC4 and PLDζ2 promote plant growth. Fig. S13 Phylogenetic and expression analysis of plant NPC and PLDζ in different species. Fig. S14 Changes in lipid levels and phosphorus levels during leaf senescence in different trees. Fig. S15 Expression of lipid and phosphorus metabolism-related genes during leaf senescence of rapeseed. Fig. S16 Proposed membrane lipids remodeling and recycling of phosphorus and carbon pathways in plants response to senescence.

13059_2024_3348_MOESM2_ESM.xlsx

Additional file 2: Table S1. The levels of lipids during leaf senescence Table S2 Mfuzz clustering showing gene expression changes in DEGs that are categorized into 8 clusters. Table S3 Gene Ontology enrichment analysis of different clusters. Table S4 Expression of lipid and phosphorus metabolism-related genes during leaf senescence of rapeseed. Table S5 Correlation of NPC4 and PLDζ2 expression levels with lipid levels during leaf senescence. Table S6 Primers used in this study.

Additional file 3: Review history.

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Yang, B., Tan, Z., Yan, J. et al. Phospholipase-mediated phosphate recycling during plant leaf senescence. Genome Biol 25, 199 (2024). https://doi.org/10.1186/s13059-024-03348-x

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Keywords

Genome Biology

ISSN: 1474-760X

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