AtSPX1-mediated transcriptional regulation during leaf senescence in Arabidopsis thaliana

Plant Science 283 (2019) 238–246

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AtSPX1-mediated transcriptional regulation during leaf senescence in Arabidopsis thaliana

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Hengyu Yan, Minghao Sheng, Chunchao Wang, Yue Liu, Jiaotong Yang, Fengxia Liu, ⁎ ⁎ Wenying Xu , Zhen Su State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China

A R T I C LE I N FO

A B S T R A C T

Keywords: AtSPX1 Leaf senescence Pi starvation response SA signal pathways Crosstalk Arabidopsis

Leaf senescence is the final stage of leaf growth, a highly coordinated and complicated process. Phosphorus as an essential macronutrient for plant growth is remobilized from senescing leaves to other vigorous parts of the plant. In this study, through data mining, we found some phosphate starvation induced genes such as AtSPX1, were significantly induced in aging leaves in Arabidopsis. We applied a reverse genetics approach to investigate the phenotypes of transgenic plants and mutant plants, and the results showed that the overexpression of AtSPX1 accelerated leaf senescence, suppressed Pi accumulation, promoted SA production and H2O2 levels in leaves, while the mutant lines of AtSPX1 showed slightly delayed leaf senescence. We conducted RNA-seq-based transcriptome analysis together with GO and GSEA enrichment analyses for transgenic vs. wild-type plants to elucidate the possible underlying regulatory mechanism. The 558 genes that were up-regulated in the overexpression plants 35S::AtSPX1/WT, were significantly enriched in the process of leaf senescence, Pi starvation responses and SA signaling pathways, as were the target genes of some transcription factors such as WRKYs and NACs. In a word, we characterized AtSPX1 as a key regulator, which mediated the crosstalks among leaf senescence, Pi starvation and SA signaling pathways in Arabidopsis thaliana.

1. Introduction Leaf senescence is a common developmental stage in plants, representing the end of the life cycle of the leaf. As a final developmental stage, leaf senescence involves the dismantling of old leaves and translocation of nutrients that are stored in them to other vigorous parts of the plant, such as young leaves, growing fruits, and seeds, a process that is very important for the growth of the whole plant. Leaf senescence is governed by developmental age and involves genetic, molecular and metabolic processes. Leaf senescence is controlled by a highly ordered transcriptional network and involves the coordination of different programmes [1–4]. This complicated process can also be induced by a series of internal and external environmental signals [5]. The external environmental signals include some adverse environments, such as nutrient limitation, low or high temperature, dark stress, drought and oxidative stress, among others. The internal factors include various phytohormones, such as abscisic acid (ABA), jasmonic acid (JA), ethylene, and salicylic acid (SA) [6,7]. Phosphorus (P) as an essential macronutrient for plant growth is remobilized from senescing leaves to growing leaves at the vegetative

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stage as well as to seeds during the reproductive stage. As a constituent of many key molecules such as adenosine triphosphate (ATP), nucleic acids, nucleoproteins, phospholipids and enzymes, phosphorus is involved in almost all metabolic processes [8,9]. The supply of P is a major limiting factor for plant growth. Phosphorus levels are abundant on the planet, but plants can only absorb P as inorganic phosphate (Pi). Thus, plants possess adaptive phosphate starvation responses (PSRs) to increase low Pi availability [10]. A promising strategy is to improve the efficiency of Pi remobilization from senescing leaves to younger sink organs. During the process of leaf senescence, the total P content in old leaves of Arabidopsis decreases by 75% [9]. Some aging-related factors have been shown to play important roles in the regulation of PSRs. For example, WRKY6 is strongly induced in aged leaves and can positively regulate leaf senescence by modulating some aging-related genes, such as the senescence-induced receptor kinase SIRK [11]. WRKY6 can also modulate the expression of PHO1, a phosphate transporter, under low phosphorus conditions [12]. Many NAC transcription factors have been reported to be highly expressed in senescent leaves [13] and up-regulated under Pi deficiency, as well as in response to ABA and osmotic treatments (e.g., drought, salt, and mannitol) [14,15], suggesting that

Corresponding authors. E-mail addresses: [email protected] (W. Xu), [email protected] (Z. Su).

https://doi.org/10.1016/j.plantsci.2019.03.005 Received 9 January 2019; Received in revised form 26 February 2019; Accepted 11 March 2019 Available online 15 March 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved.

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mechanism. To summarize, we characterized AtSPX1 as one of regulators mediating the crosstalk among leaf senescence, Pi starvation and SA signaling pathways in Arabidopsis thaliana.

some NAC transcription factor genes may be involved in the crosstalk between age-triggered leaf senescence and environmental cues, such as Pi and water status, among others. For example, ORE1/ANAC092 is one of well-characterized senescence-related NAC transcription factors in Arabidopsis. Overexpression of ORE1 results in a premature leaf senescence phenotype [16]. The ORE1 gene is highly induced by Pi deprivation [17]. In addition, ORE1 directly regulates the expression of AtBFN1, which plays an important role in the process of P recycling during leaf senescence [9]. SA is recognized as an endogenous signal, mediating plant defence against pathogens. The internal SA level in leaves gradually increases with the age of plants. SA signaling pathways play essential roles in the control of gene expression during leaf senescence [18,19]. Many leaf senescence-related genes were up-regulated after exogenous SA treatment. Mutants defective in the SA signal pathway and SA biosynthesis (npr1 and pad4 mutants) show an altered senescence phenotype [20–22]. WRKY75 transcription is induced by age, SA and other factors, and WRKY75 promotes SA production by inducing SID2. Overexpression of WRKY75 accelerates leaf senescence by promoting SA biosynthesis. Thus, WRKY75 is positive regulator of leaf senescence [6]. An interconnection between SA signals and PSRs might be present during leaf senescence. WRKY75 was identified as a positive regulator of several phosphate starvation-induced genes (PSIs), including phosphatases, Mt4/TPS1-like genes and high affinity Pi transporters [23]. Silencing of WRKY75 caused an increase in Pi content in plants [24]. Furthermore, PHR1, a master regulator of PSRs, was recently discovered to directly regulate the expression of many SA signal transduction pathway genes [10]. Overexpression of PHR1 in Arabidopsis causes an increase in P content in shoots and activates numerous PSIs, such as genes encoding the Pi transporter, phosphatase and RNase [25]. PSRs are mainly controlled by PHR1 and PHL1. Arabidopsis SPX (Syg1, Pho81 and XPR1) domain proteins: AtSPX1 and AtSPX2 have been reported as Pi-dependent repressors of PHR1 [26,27]. Over-expression of AtSPX1 increases the expression levels of some PSIs, such as ACP5, RNS1 and PAP2 [27]. Many transcriptomic studies have greatly contributed to our understanding of the fundamental mechanisms underlying leaf senescence on a genome-wide scale, including the identification of leaf senescencerelated factors and coordination of different programmes during leaf senescence [1,4,7]. A comparative analysis of the transcriptome between natural leaf senescence and dark-induced leaf senescence revealed the difference in nitrogen mobilization pathways and activation of hormone signaling pathways (SA for natural leaf senescence, JA and ethylene for dark-induced leaf senescence) between two types of leaf senescence [28]. The natural leaf senescence and salt-induced senescence were discovered to share H2O2 signaling pathways [29]. A more recent study has shown significant changes in transcription levels at different stages of leaf senescence. The results demonstrated that the major change in gene expression occurred before visible aging, and the temporal change in the gene expression level was positively related to a gain or loss of H3K4me3 and H3K9ac [2]. The transcriptomic data analysis also showed an extensive overlap of the differentially expressed genes (DEGs) in response to leaf senescence and Pi-starvation, including genes encoding Pi transporters, phospholipases, RNase and PAP isozymes [9]. This result supported a potential crosstalk between leaf senescence and Pi starvation. Overall, the transcriptomic studies in plants have greatly improved the more comprehensive understanding of the regulatory and biochemical events that occur during leaf senescence. In this study, through the ePlant Browser [30], several Pi starvationinduced genes (AtSPX1, PDLZ2 and PS3) were found with up-regulation in senescent leaves, and validated by real-time RT-PCR (Fig. 1A and B and Supplementary Fig. 1). Then, we applied a reverse genetics approach and investigated the phenotypes of the transgenic plants and mutant plants of AtSPX1. We further conducted RNA-seq and bioinformatics analysis to study the possible underlying regulatory

2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana (Col-0, atspx1 mutant lines, AtSPX1-complemented lines, and AtSPX1-overexpression lines) seeds were surface sterilized and sown on half-strength Murashige and Skoog (MS) medium with 0.8% agar in Petri plates. The seeds were stratified for 3 d at 4 °C and then transferred to a conditioning chamber with a diurnal cycle of 16 h of light (22 °C) and 8 h of darkness (19 °C). The Arabidopsis seedlings were transferred to soil 10 d after germination. 2.2. Identification of the atspx1 T-DNA insertion mutant T-DNA insertional mutants: atspx1-1 (SALK_039445) and atspx1-2 (SALK_026927) were obtained from the Arabidopsis Biological Resource Center. The mutant lines: atspx1-1 and atspx1-2 both contained a T-DNA insertion in the third exon of AtSPX1. Homozygous TDNA insertion mutant plants were confirmed by two consecutive PCR assays. The first assay involved the use of two gene-specific primers: LP (SALK_039445-LP, SALK_026927-LP) and RP (SALK_039445-RP, SALK_026927-RP). The second assay use one gene-specific primer RP (SALK_039445-RP, SALK_026927-RP) and one T-DNA-specific primer (LB). All primers used in this work are listed in Supplementary Table 9. 2.3. Construction of transgenic Arabidopsis lines 35S::AtSPX1/atspx1 and 35S::AtSPX1/WT To generate the genetic complemented line 35S::AtSPX1/atspx1 and overexpression line 35S::AtSPX1/WT, its ORF region was isolated by PCR using the forward primer 5'-GCTCTAGAATGAAGTTTGGTAAGAG TCTC-3' and reverse primer 5'-GGGGTACCCTATTTGGCTTCTTGCTCC3'. The generated fragment was inserted into the super-1300 vector in the XbaI and KpnI sites under the control of a constitutive cauliflower mosaic virus 35S promoter. The construct was verified by sequencing and introduced into the Agrobacterium tumefaciens GV3101 strain. The Arabidopsis plants were transformed using the floral infiltration method [31]. Transgenic plants were selected by hygromycin resistance and confirmed by PCR. The homozygous T2 seeds of transgenic plants were used for further analysis. 2.4. Chlorophyll content, ion leakage, H2O2 content and total P measurement For chlorophyll content measurement, the leaves were incubated in 80% acetone (v/v) 3–6 h in the dark at 4 °C. Then centrifuge for 3 min at 2700× g. Absorbance was measured at 646 and 663 nm, and the chlorophyll contents were calculated as follows: Chlorophyll a (mg/ mL) = 12.21 A663 - 2.81 A646, Chlorophyll b (mg/mL) = 20.13 A646 5.03 A663, Chlorophyll content (mg/mL) = Chlorophyll a + Chlorophyll b. Membrane ion leakage was determined by measuring electrolytes that had leaked from leaves. The third and fourth rosette leaves from plants were immersed for 3 h in 3 ml of 400 mM mannitol at 23 °C, with gentle shaking, after which the initial conductivity was recorded. Total conductivity was determined after boiling for 10 min. Conductivity was expressed as the percentage of initial conductivity versus the total conductivity [32]. Quantitative measurement of H2O2 production was performed using an Amples Red H2O2/peroxidase assay kit (Molecular Probes) according to the manufacturer's instructions [33]. For P content measurements, rosette leaves of Arabidopsis plants 239

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Fig. 1. Phenotypic analysis of the atspx1 mutant, WT, AtSPX1-complement line and AtSPX1-overexpression line during leaf senescence. A. Visualization of the expression levels of AtSPX1 in different stages of leaf development. B. qRT-PCR validation for the expression levels of AtSPX1 during leaf senescence. 35 day, fully expanded mature rosette leaves; 42 day and 47 day, early senescent leaves, leaves begin yellowing; 55 day, late senescent leaves, with 50% of leave area yellowing. C. T-DNA insertion position of the atspx1 mutant line (SALK_039445). D. Constructs used for transformation of the AtSPX1-complement and -overexpression lines. E. qRT-PCR analysis of the AtSPX1 expression levels in rosette leaves of atspx1, WT, 35S::ATSPX1/atspx1 (35S::ATSPX1/atspx1-1) and 35S::ATSPX1/WT (35S::ATSPX1/ WT-1). F. Age-dependent senescence phenotype of 6-week-old atspx1, WT, 35S::ATSPX1/atspx1 and 35S::ATSPX1/WT. The length of the white lines in the photographs represents 1 cm. G. Rosette leaves of 6-week-old atspx1, WT, 35S::ATSPX1/atspx1 and 35S::ATSPX1/WT. Bar = 1 cm. H. Chlorophyll content in 6-week-old rosette leaves of atspx1, WT, 35S::AtSPX1/atspx1 and 35S::AtSPX1/WT. I. Membrane ion leakage in 6-week-old rosette leaves of atspx1, WT, 35S::AtSPX1/atspx1 and 35S::AtSPX1/WT. J. Total P content in 6-week-old rosette leaves of atspx1, WT, 35S::AtSPX1/atspx1 and 35S::AtSPX1/WT. Significant difference between the mutant, transgenic line and WT was determined according to Student’s t-test. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001. The one-way ANOVA results for genotypic variation comparisons in H–J were listed in Supplementary Table 1.

grown on soil were harvested. The samples were oven-dried at 80 °C to a constant weight, ashed at 300 °C for 1 h and 575 °C for 5 h in a muffle furnace, and then dissolved in 0.1 M HCl. The total P content in the

samples was quantified as described previously [34]. The one-way ANOVA was applied to test the significant genotypic variation, and the student’s t-test was used to compare significant 240

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differences between transgenic lines and WT plants.

2.8. Real-time RT-PCR

2.5. Plant hormone content measurement

Reverse transcription was performed using Moloney Murine Leukaemia Virus (M-MLV; Invitrogen). 10 μl samples containing 2 μg of total RNA, and 20 pmol of random hexamers (Invitrogen) was heated at 70 °C for 2 min, and then chilled on ice for 2 min. We added reaction buffer and M-MLV to a total volume of 20 μl containing 200 units of MMLV, 20 pmol random hexamers, 500 μM dNTPs, 50 mM Tris−HCl (pH 8.3), 3 mM MgCl2, 75 mM KCl and 5 mM dithiothreitol. The samples were then heated at 37 °C for 1.5 h [41]. The cDNA samples were diluted to 2 ng/μl for real-time RT-PCR. Triplicate quantitative assays were performed using 8 ng of cDNA with SYBR Green Master mix and an ECO Real-Time PCR system (Illumina) according to the manufacturer’s protocol. Primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and were listed in Supplementary Table 9. The relative quantitation method (ΔΔCT) was used to evaluate the quantitative variation among replicates. The 18S rRNA was applied as an internal control to normalize all data for the real-time RT-PCR experiments.

Rosette leaves of Arabidopsis plants grown on soil were harvested. Fifty microliters of the internal standard working solution and 500 μl of the extract were separately added to the 50 mg sample, vortexed and then shaken at 4 °C for 30 min. Then, 1 ml of the extract CHCL3 was added, followed by vortexing and shaking at 4 °C for 30 min. After centrifugation, the bottom layer of liquid was collected and dried with helium at room temperature. The samples were reconstituted with 0.1 ml of MeOH and then filtered with a 0.1 μM filter. They were then subjected to ultra-performance liquid chromatograpUhy (UPLC) analysis. The plant samples were analysed using an UPLC-HRMS system (UPLC, Waters ACQUITY UPLC i-Class, Milford, MA, USA; MS, Thermo Fisher Q-Exactive, Bremen, Germany) equipped with a heated electrospray ionization (HESI) source. Chromatographic separation was performed on a Poroshell 120 EC-C18 column (3.0 × 75 mm, 2.7 μm, Agilent, USA) at a flow rate of 0.4 ml min−1. The mobile phases consisted of 0.05% AA in water (phase A) and 0.05% AA in ACN (phase B) for the analysis of SA, ABA, IAA and JA. For the detection of ZT and OPDA, 0.1% FA in water (phase A) and 0.1% FA in MeOH (phase B) were used. The gradient programme was set as follows: 90% A at 0 min to 60% A at 6.25 min, 10% A at 7.5 min and holding for 3 min, followed by a return to the initial condition. The column temperature was 35 °C. MS analysis was performed in both positive and negative ion modes. The HESI source parameters were set as follows: spray voltage (+) at 3.5 kV, spray voltage (-) at 3 kV, capillary temperature at 320 °C, sheath gas flow rate at 30 arbitrary units, aux gas flow rate at 10 units, sweep gas flow rate at 5 units, gas heater temperature at 350 °C and S-lens RF level at 55%. The full MS scan (resolution: 70,000, AGC target: 3E6, maximum IT: 100 ms, scan range: 50–750 m/z) combined with the targeted MS2 scan (resolution: 17,500, AGC target: 2E5, maximum IT: 100 ms, isolation window: 2.0 m/z) was performed for data acquisition.

2.9. Motif analysis The 2-kb promoter sequences of genes, which were up-regulated in 35S::AtSPX1/WT, were extracted. Motifs from the AthaMap webserver [42], Plant Cis-acting Regulatory DNA Elements database [43] and PlantCARE database [44] were collected for the enrichment analysis. The algorithm used to calculate significance has been described previously [45]. Motifs with a P-value less than 0.05 were considered significantly enriched. 3. Results and discussion 3.1. Reverse genetic analysis revealed AtSPX1 as a positive regulator during natural leaf senescence Leaf senescence is the final stage of leaf development, usually accompanied by the activation of a series of metabolic processes. During the process of leaf senescence, the total P content in old leaves of Arabidopsis decreases by 75% [9]. Using the ePlant Browser [30], we found the Pi-sensing gene AtSPX1 significantly highly expressed in old leaves (Fig. 1A), which was validated by real-time RT-PCR (Fig. 1B). To investigate the roles of AtSPX1 during leaf senescence, we performed a genetic analysis of age-triggered leaf senescence phenotypes for the SALK T-DNA insertion line atspx1 (atspx1-1, SALK_039445) (Fig. 1C), AtSPX1-complemented lines, AtSPX1-overexpression lines and wild-type (WT). The cloned full-length CDS for AtSPX1 was used to generate two types of transgenic lines: 35S::AtSPX1/atspx1 to complement the AtSPX1 mutation in the atspx1 mutant, and 35S::AtSPX1/WT to overexpress the AtSPX1 gene in WT (Fig. 1D). Three AtSPX1-overexpression lines and three AtSPX1-complemented lines were obtained. The transgenic lines were validated by real-time RT-PCR (Fig. 1E and Supplementary Fig. 2C). The 35S::AtSPX1/WT and 35S::AtSPX1/atspx1 displayed a precocious leaf senescence phenotype with more yellow leaves, while the mutant atspx1 exhibited a slightly delayed leaf senescence phenotype compared with WT (Fig. 1F and G and Supplementary Fig. 2A and B). In addition, we performed phenotype analysis for another T-DNA insertion line of AtSPX1 (atspx1-2, SALK026927) which has a T-DNA insert in the third exon of AtSPX1. atspx1-2 also exhibited slightly delayed leaf senescence phenotype than WT (Supplementary Fig. 3A,B). After flowering, the premature aging phenotype of 35S::AtSPX1/WT and 35S::AtSPX1/atspx1 is more obvious (Supplementary Fig. 4A,B). Some senescence-related parameters were determined. The 35S::AtSPX1/WT and 35S::AtSPX1/atspx1 showed a significant decrease in chlorophyll content, while mutant line atspx1 showed a higher chlorophyll content compared with WT (Fig. 1H, Supplementary

2.6. RNA extraction Rosette leaves of Arabidopsis plants grown in soil were sampled for RNA extraction. The rosette leaves (Col-0, atspx1 mutant line, and AtSPX1-overexpression line 35S::AtSPX1/WT, as well as other transgenic lines or time points) were homogenized in liquid nitrogen before RNA isolation. Total RNA was isolated using TRIZOL® reagent (Invitrogen, CA, USA) and purified using Qiagen RNeasy columns (Qiagen, Hilden, Germany). 2.7. RNA-seq analysis Sequencing libraries were constructed by the Beijing Genomics Institute and sequenced using an IlluminaHiSeq™ 2000 following standard protocols. The paired-end mRNA-seq consisted of read lengths of 150 bp for each sample. RNA-seq data with 3 biological replicates were generated. Sequencing reads were aligned to the reference genome (TAIR10) by TopHat v2.0.9 [35] software using default parameters. The total reads, mapped reads and mapping rate were listed in Supplement Table 2. Read counts for each gene were calculated using summarizeOverlaps (R package GenomicAlignments) with the following setting: mode = "Union", singleEnd = FALSE, ignore.strand = TRUE, fragments = TRUE [36]. DESeq2 version 1.6.3 were used to identified differential expressed genes [37]. We defined the genes that were up-regulated (or down-regulated) in 35S::AtSPX1/WT based on expression levels that were significantly higher (or lower) in 35S::AtSPX1/WT than in WT or atspx1. GO enrichment analysis was performed using agriGOv2 [38] and REVIGO [39]. Gene set enrichment analysis and transcript factor target enrichment were performed using PlantGSEA [40]. 241

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enrichment analysis (GSEA) of KEGG, the gene family for genes upregulated in 35S::AtSPX1/WT by PlantGSEA [40]. In the KEGG category, the SA biosynthesis-related pathway “Phenylpropanoid biosynthesis” [P-value = 1.19E-09] was significantly enriched. In the gene family category, “Receptor kinase-like protein family” [P-value = 3.64E-03], “WRKY Transcription Factor family” [P-value = 3.73E-03] and “AP2-EREBP Transcription Factor Family” [P-value = 6.99E-03] were enriched (Table 1). Many WRKY and NAC transcription factors were up-regulated in 35S::AtSPX1/WT (Supplementary Table 3). We further performed transcription factor target and motif enrichment analyses for the genes that were up-regulated in 35S::ATSPX1/WT. We collected some transcription factor targets from public platform [54], and we performed an enrichment analysis for the genes that were up-regulated in 35S::ATSPX1/WT. Many TF targets were enriched, including targets of WRKYs (WRKY33, WRKY40, WRKY65, WRKY50, WRKY75 and WRKY45) and NACs (NAC055, NAC083, NAC047, NAC046, NAC092), among others (Table 2). Motif analysis for the 558 genes up-regulated in 35S::AtSPX1/WT revealed that many transcription factor binding motifs were significantly enriched, including the WRKY putative binding sites W-box “TTGAC” [Z-score = 4.40; P-value = 5.5E-06] and NAC putative bind sites “CACG” [Z-score = 4.96; P-value = 4E-07], among others (Supplementary Table 5). Based on functional enrichment analysis of differentially expressed genes, several genes involved in phosphate starvation response, SA signaling transduction and leaf senescence were selected for real-time RT-PCR validation using additional replicate (Fig. 2C). The fold change of genes analysed by real-time RT-PCR were not exactly the same as those obtained in the RNA-seq data, but the change trends were similar. WRKY75 could be induced in response to Pi deprivation. WRKY75 was identified as a positive regulator of several PSIs, including phosphatases, Mt4/TPS1-like genes and high-affinity Pi transporters. WRKY75 RNAi plants showed an increased total P content [24]. Recently, WRKY75 was identified as a positive regulator of leaf senescence in Arabidopsis thaliana [6]. WRKY75 was reported to promote SA production by inducing the transcription of SID2 and to suppress scavenging, partly by repressing the transcription of CATALASE2 (CAT2). Mutant of NAC055 show delayed leaf senescence phonotype [55]. Some SA responsive genes such as PR1, PR4, DLO1 were highly expressed in 35S::AtSPX1/WT (Fig. 2C, Supplementary Table 3). PR1 and PR4 is an SA-induced pathogen responsive gene. DLO1 (DMR6-LIKE OXYGENASE 1) is activated in response to SA-mediated immune response for pathogen infection, also induced during senescence. DLO1 encodes an enzyme that can convert SA to 2,3-dihydroxybenzoic acid. By activating DLO1 during senescence and SA signaling, the plant can fine-tune control immune response and the rate of leaf senescence, and preventing SA over-accumulation, which adversely affects plant growth and development [21,56]. There is a interplay between SA signaling and ROS signals [57]. Some of genes involved in regulation of H2O2 metabolism were also highly expressed in 35S::AtSPX1/WT, such as HB1, GOX3 (Fig. 2C, Supplementary Table 3). GOX3 (GLYCOLATE OXIDASE 3) encodes a glycolate oxidase, and could promote the generation of H2O2 [58]. Some genes up-regulated in 35S::AtSPX1/WT such as MC2, ELI3-2, also involved in programed cell death (Fig. 2C, Supplementary Table 3). ELI3-2 (ELICITOR-ACTIVATED GENE 3-2) was induced by in response to a variety of pathogens, and involved in planttype hypersensitive response [59]. In addition, many senescence-related genes, such as SAG201, SAG12, SAG29 and YLS2 were induced in 35S::AtSPX1/WT (Supplementary Table 3). SAGs is a specific set of “senescence-associated genes”. SAG201 is a positive regulator of leaf senescence [60]. SAG29, SAG12 and YLS2 (YELLOW-LEAF-SPECIFIC GENE 2) are marker genes for leaf senescence [61]. We also compared DEGs among atspx1, WT and 35S::AtSPX1/WT. The number of common genes between genes up-regulated in 35S::AtSPX1/WT (vs. WT) and (vs. atspx1) was higher than common genes between genes up-regulated in 35S::AtSPX1/WT (vs. WT) and

Fig. 2D and Fig. 3C). Ion leakage is an indicator for the integrity of the plasma membrane. When leaves begin senescence, membrane become fragile, and electrolytes leak out of the cell [46]. Ion leakage is usually used to assess degree of senescence [47–49]. Ion leakage was higher in 35S::AtSPX1/WT and 35S::AtSPX1/atspx1 than WT, while it was decreased in atspx1-1 and atspx1-2 (Fig. 1I and Supplementary Fig. 3D). Given that AtSPX1 is a major regulator of PSRs, we measured total P content in the leaves of 35S::AtSPX1/WT, 35S::AtSPX1/atspx1, atspx1 and WT. The 35S::AtSPX1/WT and 35S::AtSPX1/atspx1 showed reduced P content, and atspx1 showed increased P content compared with WT (Fig. 1J). Thus, AtSPX1 negatively modulated the P content. Using a one-way ANOVA test, the variation of chlorophyll content, ion leakage and P content were found be significantly altered by genotypic variation (Supplementary Table 1). Overall, the genetic complementation experiments and overexpression studies verified AtSPX1 act as a positive regulator in age-triggered leaf senescence. 3.2. RNA-seq-based transcriptome analysis for transgenic vs. wild-type plants A transcriptomic strategy was used to explore the mechanism regulated by AtSPX1 for age-triggered leaf senescence. We performed RNA-seq to investigate the genome-wide gene expression differences between 35S::AtSPX1/WT, WT and atspx1. Forty-day-old rosette leaves of atspx1, WT and 35S::AtSPX1/WT were harvested for RNA-seq, with three independent biological replicates for each sample (Supplementary Table 2). Using DESeq2, we assigned differential expressed genes (DEGs) with a cut-off value log2(fold change) ≥ 1 and FDR ≤ 0.05. Finally, we identified 558 genes that were significantly up-regulated in 35S::AtSPX1/WT and 270 genes that were down-regulated in 35S::AtSPX1/WT compared with WT (Supplementary Table 3). We then conducted GO enrichment analysis for these up-regulated genes in 35S::AtSPX1/WT. The GO terms “cellular response to phosphate starvation” [GO:0016036, FDR = 2.70E-13] and “galactolipid biosynthetic process” [GO:0019375, FDR = 5.20E-15] were significantly enriched (Fig. 2A, Supplementary Table 4), including AtSPX1, AtSPX3, WRKY75, PS2, PAP22, PHT1;5, and PLDGAMMA3, among others. These genes play essential roles in the catabolism of the various P-containing biomolecule pools, such as phospholipids and phosphoanhydrides, exporting released Pi to sink tissues. The replacement of phospholipids by galactolipids in plant membranes is very important for plant growth under Pi-deprivation conditions [50]. The enriched GO terms also include “salicylic acid mediated signaling pathway” [GO:0009863, FDR = 1.50E-05], “jasmonic acid mediated signaling pathway” [GO:0009867, FDR = 6.70E-03], “salicylic acid biosynthetic process” [GO:0009697, FDR = 1.10E-02], “jasmonic acid biosynthetic process” [GO:0009695, FDR = 1.20E-05], “leaf senescence” [GO:0010150, FDR = 1.80E-02], and “regulation of hydrogen peroxide metabolic process” [GO:0010310, FDR = 8.60E-05], among others (Fig. 2A, Supplementary Table 4). In addition, genes down-regulated in 35S::AtSPX1/WT were mainly associated with “response to light stimulus” [GO:0009416, FDR = 2.50E-05], “myo-inositol hexakisphosphate (phytic acid) biosynthetic process” [GO:0010264, FDR = 1.40E-03], “ethylene biosynthetic process” [FDR = 6.80E-06], “cytokinin-activated signaling pathway” [GO:0009736, FDR = 2.50E-02], “regulation of transcription, DNA-templated” [GO:0006355, FDR = 1.40E-07] and “circadian rhythm” [GO:0007623, FDR = 1.40E-03] (Fig. 2B, Supplementary Table 4). There are several GO terms possibly with negative relation to Pi starvation response and leaf senescence. For example, Phytic acid, which could be hydrolyzed generated Pi, play an important role in phosphate homeostasis. Disruption of the kinase for phytic acid synthesis could elevate Pi uptake/allocation activities and activate a subset of Pi starvation-responsive genes [51,52]. Cytokinin is a wellestablished negative regulator of leaf senescence [53]. In addition to GO enrichment analysis, we performed a gene set 242

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Fig. 2. RNA-seq-based transcriptome data analysis. A–B. GO enrichment analysis for genes that were up-regulated (A) and down-regulated (B) in 35S::AtSPX1/ WT by agriGOv2 and REViGO. The scatter plot shows the cluster representatives in two-dimensional space derived by applying multidimensional scaling to a matrix of the significant GO terms with semantic similarities. The bubble colour and size indicate the log10FDR (legend in bottom right-hand corner). The colours from redto-green represent the significance level of the GO terms from high to low. Fisher’s exact test, P-value was adjusted using the Benjamini–Yekutieli method, FDR ≤ 0.05. C. qRT-PCR validation the expression levels of several key genes associated with PSRs, SA signaling transduction and leaf senescence in atspx1, WT and 35S::AtSPX1/WT. ANAC055–AT3G15500; WRKY75–AT5G13080; WRKY45–AT3G01970; PR1–AT2G14610; PR4–AT3G04720; DLO1–AT4G10500; MAPKKK19– AT5G67080; ATABCG40–AT1G15520; YLS2–AT3G51430; PGIP2–AT5G06870; AHB1–AT2G16060; AtMCP1c–AT4G25110; ELI3-2–AT4G37990; ATARD3– AT2G26400. Table 1 Enriched gene sets in genes up-regulated in 35S::ATSPX1/WT by PlantGSEA. Gene Set Name (No. Genes)

Category

No. Genes in Overlap

P-value

Receptor kinase-like protein family (307) WRKY Transcription Factor Family (72) AP2-EREBP Transcription Factor Family (138) Phenylpropanoid biosynthesis (104) Phenylalanine metabolism (80) Methane metabolism (82)

GFam GFam GFam KEGG KEGG KEGG

14 6 8 16 14 14

3.64E-03 3.73E-03 6.99E-03 1.19E-09 2.99E-09 3.96E-09

were significantly enriched in genes up-regulated in 35S::AtSPX1/WT (Supplementary Fig. 6A, Supplementary Table 6). GO enrichment analysis was performed for those DEGs and common genes between of them. The most enriched GO terms mainly were some hormone related

genes up-regulated in WT (vs. atspx1) (Supplementary Fig. 5, Supplementary Table 6). Further, we compared genes up-regulated in 35S::AtSPX1/WT (vs. WT and vs. atspx1) and genes up-regulated in senescent leaves, and found that genes up-regulated in senescent leaves 243

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GO terms such as “salicylic acid mediated signaling pathway”, “salicylic acid biosynthetic process”, “jasmonic acid mediated signaling pathway” and “abscisis acid mediated signaling pathway”, which were significantly enriched in all gene sets (Supplementary Fig. 6B). In addition, some of aging related GO terms such “aging”, “senescence”, “regulation of hydrogen peroxide metabolic process”, and “programmed cell death”, were also enriched in almost all the groups (Supplementary Fig. 6B). GO terms of “Phosphorus starvation response” were enriched in all groups related to the genes up-regulated in 35S::AtSPX1/WT (Supplementary Fig. 6B). The result further indicated that AtSPX1 might function as one of important regulators in leaf senescence, Pi starvation response and SA signaling pathway. Further, we compared the genes up-regulated in senescent leaves with those in response to Pi-deprivation [62] and Benzothiadiazole (BTH, SA analogue) treatment [63]. About 14.9% (217/1449) and 19.9% (288/1449) genes up-regulated in senescent leaves were induced in response to Pi-starvation and BTH treatment, respectively (Supplementary Fig. 7), which included SA pathways related genes: WRKY75, PR1, PR4, PR5, DLO1; Pi-starvation induced genes: PHT3;2 (PHOSPHATE TRANSPORTER 3;2), PLDP2 (PHOSPHOLIPASE D P2), ATIPS2 (INDUCED BY PI STARVATION 2) and leaf senescence related genes: SAG13, DIN2, SRG1, YLS9 (Supplementary Table 7). This indicated the relevance among leaf senescence, Pi starvation response and SA signaling pathways. In addition, some genes up-regulated in senescent leaves and stress response such as Pi starvation and SA signaling, were up-regulated in 35S::AtSPX1/WT, such as WRKY75, PR1, PR4, DLO1, PLDP2, ATIPS2, SRG1 and DIN2, which further suggested that AtSPX1 might act as a regulator mediated the crosstalk of SA signal transduction, Pi starvation responses and leaf senescence (Supplementary Table 7).

Table 2 Transcription factor targets enrichment analysis for genes up-regulated in 35S::AtSPX1/WT. Gene Set Name (No. Genes)

Gene family

No. Genes in Overlap

FDR

WRKY33 target genes (2104) PHR1 target genes (2364) WRKY70 target genes (6714) * ANAC055 target genes (6370) PDF2 target genes (1978) WRKY40 target genes (1478) EIN3 target genes (1314) PIF4 target genes (7338) WRKY65 target genes (7252) WRKY50 target genes (10181) AtHB32 target genes (12176) WRKY24 target genes (6051) * WRKY75 target genes (13330) PHL1 target genes (4077) NAP target genes (4061) WRKY18 target genes (1290) NAM target genes (4383) WRKY55 target genes (7217) ANAC038 target genes (5259) WRKY22 target genes (14815) WRKY14 target genes (7095) * ATHB24 target genes (10305) * WRKY45 target genes (7776) WRKY25 target genes (4889) WRKY30 target genes (3839) PHL4 target genes (5286) ATHB23 target genes (12750) WRKY15 target genes (14748) MYB3R1 target genes (7322) ANAC046 target genes (4439) ATHB25 target genes (13777) ATHB34 target genes (13948) * ANAC047 target genes (6448) ANAC083 target genes (8891) WRKY71 target genes (7206) ANAC087 target genes (2085) ANAC092 target genes (6319) CUC3 target genes (5263) ATHB5 target genes (14295) WRKY27 target genes (13723) WRKY8 target genes (14021) ANAC058 target genes (9742) WRKY31 target genes (3122) ATHB33 target genes (12208) ATAF1 target genes (7615) WRKY29 target genes (11906) WRKY6 target genes (4646)

WRKY G2-like WRKY NAC HD-ZIP WRKY EIL bHLH WRKY WRKY ZF-HD WRKY WRKY G2-like NAC WRKY NAC WRKY NAC WRKY WRKY ZF-HD WRKY WRKY WRKY G2-like ZF-HD WRKY MYB NAC ZF-HD ZF-HD NAC NAC WRKY NAC NAC NAC HD-ZIP WRKY WRKY NAC WRKY ZF-HD NAC WRKY WRKY

112 110 216 202 90 75 69 221 219 281 319 189 340 141 140 64 146 211 166 363 206 273 219 154 127 161 318 356 204 139 335 338 184 236 200 79 180 156 342 331 336 251 104 300 205 293 139

5.25E-18 4.58E-14 9.94E-13 4.65E-11 8.45E-11 8.45E-11 1.71E-10 1.91E-10 1.91E-10 2.47E-10 8.09E-10 9.76E-10 1.73E-09 1.96E-09 2.86E-09 6.50E-09 8.55E-09 8.76E-09 1.26E-08 2.14E-08 2.84E-08 4.68E-08 7.10E-08 7.93E-08 2.59E-07 2.78E-07 2.91E-07 3.26E-07 8.58E-07 8.75E-07 9.71E-07 1.09E-06 1.20E-06 1.53E-06 1.55E-06 1.98E-06 1.98E-06 2.28E-06 3.32E-06 3.32E-06 4.52E-06 4.57E-06 4.84E-06 6.05E-06 8.49E-06 9.36E-06 9.39E-06

3.3. AtSPX1 positively modulates SA and H2O2 accumulation during leaf senescence RNA-seq-based transcriptome analysis revealed that SA biosynthesis, SA signal transduction and H2O2 metabolism-related biological processes were significantly enriched in the genes that were up-regulated in 35S::AtSPX1/WT (Fig. 3A). Phytohormones have long been known to affect the timing of leaf senescence [3,5,7]. Thus, we measured the content of several hormones: SA, JA, IAA and ABA in the leaves of 35S::AtSPX1/WT, atspx1 and WT. The SA content in 35S::AtSPX1/WT leaves was approximately 5- and 8-fold higher than those in WT and atspx1, respectively (Fig. 3A). The 35S::ATSPX1/WT and WT accumulated more JA than atspx1, and there was no difference in the content of JA between 35S::AtSPX1/WT-1 and WT (Fig. 3A). The content of two other hormones, ABA and IAA, did not show obvious differences among 35S::ATSPX1/WT, atspx1 and WT (Fig. 3A). These

* these TFs were up-regulated in 35S::AtSPX1/WT.

Fig. 3. Hormone content and H2O2 content measurement. (A) SA, JA, ABA and IAA content in leaves of 5-week-old atspx1, WT and 35S::ATSPX1/WT. (B) H2O2 content in leaves of 5-week-old atspx1, WT and 35S::ATSPX1/ WT. Data were represented as the means ± sd, n = 3. Significant difference between the mutant, transgenic line and WT were determined according to Student’s t-test. *P-value ≤ 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001. The one-way ANOVA results for genotypic variation comparisons in A–B are listed in Supplementary Table 1.

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regulation of some key senescence-related transcription factor genes, such as WRKYs (WRKY75, WRKY45) and NACs (NAC047, NAC055), further causing SA accumulation, increased H2O2 levels, and Pi starvation, ultimately accelerating the leaf senescence process. WRKY45 has been characterized an important regulator in modulating PSRs and leaf senescence [69,70]. WRKY75 has been reported to be involved in the regulation of Pi starvation responses, SA synthesis, H2O2 metabolism and leaf senescence [6,24]. WRKY75, WRKY45 and their downstream genes, which are mostly positively regulated by AtSPX1, are significantly enriched in Pi starvation, leaf senescence and the SA response (Supplementary Table 8). NAC047 and NAC055 are involved in the process of leaf senescence mediated by EIN2 [71]. Many target genes of NAC047, NAC055 themselves were positively regulated by AtSPX1 (Supplementary Table 8). In addition, many target genes of those transcription factors were also enriched in senescence, SA signal transduction and Pi starvation responses (Supplementary Fig. 8). Overall, the genetic and transcriptome analyses suggest that AtSPX1 plays an essential role in regulating the age-dependent leaf senescence process relevant to many physiological processes. Acknowledgements We thank Qunlian Zhang for technical support. This work was supported by grants from the National Natural Science Foundation of China [31771467, 31571360, 31371291]. The authors declare that they have no conflicts of interest.

Fig. 4. Working model for the role of AtSPX1 in age-triggered leaf senescence. With leaf senescence, AtSPX1 expression levels was increased, which induced the expression of some key transcription factors WRKYs (WRKY75, WRKY45) and NACs (NAC047, NAC055), these transcription factors regulate some genes associated with phosphate starvation responses, SA signal transduction, H2O2 metabolism and leaf senescence, which results in the decrease of P content, accumulation of SA and H2O2, ultimately accelerating the leaf senescence process.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2019.03.005. References

results showed that over-expression of AtSPX1 promoted SA production, which indicated that SA might plays a major role in AtSPX1mediated age-triggered leaf senescence. During leaf senescence, endogenous accumulated SA is important for the development of senescence phenotype [6]. SA can affect the expression of a series of aging-related genes, including SAG21, SRG1, WRKY6, etc. A mutant of SA biosynthesis, NahG, with low SA levels shows a delayed senescence phenotype [64]. Genes related to SA signal transduction, such as PR3, PR1, WRKY62, MES9 and HB1, were upregulated in 35S::AtSPX1/WT (Supplementary Table 3). H2O2 plays an important role in the aging process, which can also be induced by SA [65,66]. H2O2 content was determined by using the H2O2 quantitative assay. The 35S::AtSPX1/WT produced more H2O2 than WT and atspx1. The mutant atspx1 had the lowest H2O2 levels (Fig. 3B). Using one-way ANOVA, the genotypic variation (mutation and overexpression of AtSPX1 gene) was found to significantly alter the H2O2 levels (Supplementary Table 1). Thus, over-expression of AtSPX1 promoted H2O2 levels in leaves. Exogenous application of H2O2 can also promote premature leaf aging, and the decline in chlorophyll and soluble protein is associated with increased levels of H2O2 [67]. In addition, SA treatment can also promote the production of H2O2 [68]. The phenotypic and RNA-seq data analysis results revealed that AtSPX1 might positively regulate age-triggered leaf senescence by affecting the SA immunity pathway, H2O2 metabolism and phosphate starvation genes.

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