Abstract
Background
Phosphorus (P) is an essential macronutrient for plant growth that participates in a series of biological processes. Thus, P deficiency limits crop growth and yield. Although Stylosanthes guianensis (stylo) is an important tropical legume that displays adaptation to low phosphate (Pi) availability, its adaptive mechanisms remain largely unknown.
Results
In this study, differences in low-P stress tolerance were investigated using two stylo cultivars (‘RY2’ and ‘RY5’) that were grown in hydroponics. Results showed that cultivar RY2 was better adapted to Pi starvation than RY5, as reflected by lower values of relative decrease rates of growth parameters than RY5 at low-P stress, especially for the reduction of shoot and root dry weight. Furthermore, RY2 exhibited higher P acquisition efficiency than RY5 under the same P treatment, although P utilization efficiency was similar between the two cultivars. In addition, better root growth performance and higher leaf and root APase activities were observed with RY2 compared to RY5. Subsequent RNA-seq analysis revealed 8,348 genes that were differentially expressed under P deficient and sufficient conditions in RY2 roots, with many Pi starvation regulated genes associated with P metabolic process, protein modification process, transport and other metabolic processes. A group of differentially expressed genes (DEGs) involved in Pi uptake and Pi homeostasis were identified, such as genes encoding Pi transporter (PT), purple acid phosphatase (PAP), and multidrug and toxin extrusion (MATE). Furthermore, a variety of genes related to transcription factors and regulators involved in Pi signaling, including genes belonging to the PHOSPHATE STARVATION RESPONSE 1-like (PHR1), WRKY and the SYG1/PHO81/XPR1 (SPX) domain, were also regulated by P deficiency in stylo roots.
Conclusions
This study reveals the possible mechanisms underlying the adaptation of stylo to P deficiency. The low-P tolerance in stylo is probably manifested through regulation of root growth, Pi acquisition and cellular Pi homeostasis as well as Pi signaling pathway. The identified genes involved in low-P tolerance can be potentially used to design the breeding strategy for develo** P-efficient stylo cultivars to grow on acid soils in the tropics.
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Background
Phosphorus (P) is one of the essential macronutrients for plant growth and development. P is involved in the processes of photosynthesis, respiration, energy metabolism and signal transduction in plants [1]. Furthermore, P is also an important structural component of various biomolecules in plant cells, including adenosine triphosphate (ATP), phospholipids, DNA and RNA [2]. Although total P is abundant in soils, P is easily immobilized by soil components into unavailable forms that cannot be directly utilized by plants [3]. Thus, low phosphate (Pi) availability is considered a major limiting factor for crop growth, especially in acid soils that occupy about 50% of the world’s arable land [3, 4]. To obtain high yields in traditional agricultural systems, farmers need to apply excessive quantities of P fertilizer, potentially leading to soil deterioration and eutrophication problems [5]. Furthermore, P fertilizer is derived from mined phosphate rock, which is a finite resource that is slowly depleting [6]. Therefore, improving the absorption and utilization of soil P can be an effective way for increasing crop yield and reducing fertilizer P application. Such improvements aim for the development of a more sustainable and environmentally sound agriculture.
To cope with low-P stress, plants have improved Pi uptake and homeostasis through a wide range of morphological, physiological and molecular changes, such as modifying root morphology and architecture, increasing secretion of organic acid and acid phosphatases, enhancing expression of high-affinity Pi transporters, develo** symbioses with arbuscular mycorrhizal fungi, and regulating complex P signaling networks in plant cells [7, 8]. It has been well demonstrated that plants display plasticity in root growth under P deficiency by changing root morphology and architecture, and thus increasing acquisition of P from the soil [9, 10]. For example, increase in root length and root/shoot ratio is observed in maize (Zea mays) [11], faba bean (Vicia faba) [12], rapeseed (Brassica napus) and wheat (Triticum aestivum) [13] in response to low P supply. Furthermore, acid phosphatase activities are up-regulated by Pi deprivation in many plants, such as rice (Oryza sativa), soybean (Glycine max) and chickpea (Cicer arietinum), which could contribute to increased organic P utilization [14,15,16].
To date, a variety of P responsive genes have been identified to participate in Pi uptake and homeostasis [8, 17]. For example, PHOSPHATE STARVATION RESPONSE 1 (AtPHR1) in Arabidopsis and OsPHR2 in rice, encoding the MYB transcription factor, are the central regulators involved in Pi signaling pathway [8]. AtPHR1 is demonstrated to regulate a set of Pi starvation induced (PSI) genes through binding to the P1BS cis element of target genes [18]. Furthermore, a negative regulatory role for protein containing the SYG1/PHO81/XPR1 (SPX) domain in rice is documented where OsSPX1 suppresses the transcripts of several PSI genes, such as Pi transporters (OsPT2 and OsPT6) and purple acid phosphatases (OsPAP10) [7]. A group of Pi transporters have been functionally characterized to be involved in Pi uptake and/or translocation in many plants; examples include: AtPT1 and AtPT2 from Arabidopsis [19, 20], OsPT1/9/10 from rice [21, 22] and GmPT5/7 from soybean [23, 24]. In addition, numerous purple acid phosphatase (PAP) homologues have also been demonstrated to function in Pi release from organic P, including AtPAP10/12/26 from Arabidopsis [25], OsPHY1 from rice [ In this study, two stylo (Stylosanthes guianensis) cultivars, ‘RY2’ and ‘RY5’, were used, which were widely grown in South China [33]. The stylo seeds were provided by the Institute of Tropical Crop Genetic Resources (TCGRI), Chinese Academy of Tropical Agricultural Sciences (CATAS), Hainan, China. Experiments were performed in a greenhouse at temperatures of 25 °C to 32 °C under natural sunlight with a photoperiod of about 13 h at the TCGRI, CATAS, Hainan, China (19°30′N, 109°30′E). Seeds were germinated for 3 d, and stylo seedlings were then transferred to a modified Hoagland nutrient solution containing 250 μM KH2PO4 for 14 d as previously described [71]. After that, seedlings were separately transplanted into nutrient solution supplied with 0, 100 and 250 μM KH2PO4, which were regarded as low (Pi deprivation), moderate and high P supply treatments, respectively. The nutrient solution was adjusted to a pH value of 5.8 and refreshed weekly. After 21 d of P treatments, shoots and roots were separately harvested for further analysis. An individual hydroponic box containing three seedlings of each stylo cultivar was set as one biological replicate. Each treatment included three biological replicates. Plant fresh roots were scanned using an Epson 12000XL scanner (Epson, Japan) with a resolution of 300 dpi, and the obtained image was saved as JEPG format. Total root length, root surface area and root volume were analyzed with WinRhizo Pro software (Regent Instruments Inc., Quebec, Canada). After that, shoot and root samples were oven dried at 75 °C for 7 d, and the dry mass of shoots and roots was further determined. For P concentration analysis, approximately 0.07 g dry samples were burned to ash at 600 °C in a muffle furnace. The sample of ash was absolutely dissolved in 100 mM HCl, and the supernatant was then used for P concentration analysis as previously described [72]. APase activities in stylo leaf and root were analyzed as previously described [38] with some modification. Approximately 0.15 g of leaf and root samples were ground in 1.5 mL of 45 mM Na-acetate buffer (pH 5.0) at 4 °C. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants were mixed with 2 mL of 45 mM Na-acetate buffer (pH 5.0) containing 1 mM ρ-nitrophenyl phosphate (Sigma, Saint Louis, MO, USA). After incubation at 37 °C for 15 min, the reaction was terminated by the addition of 1 mL of 1 M NaOH. APase activity was spectrophotometrically detected at 405 nm and expressed as micromoles of ρ-nitrophenyl phosphate hydrolyzed per mg protein per min. Protein concentration in the extracts was analyzed using the Coomassie Brilliant Blue staining method [73]. Total RNA from roots of RY2 at 0 (LP) and 250 (HP) μM KH2PO4 treatments was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. RNA purity and integrity were assessed by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 (Agilent Technologies, Palo Alto, CA, USA), respectively. RNA sequencing analysis was conducted by Novogene Bioinformatics Technology Co., Ltd. (Bei**g, China). RNA-seq libraries were constructed using the NEBNext® UltraTM RNA Library Prep kit (NEB, Beverly, MA, USA), and the cDNA libraries were sequenced using an Illumina HiseqTM platform (Illumina, San Diego, CA, USA). The 150-bp paired-end reads (PE150) were generated. RNA-seq raw data were obtained using the Casava v.1.8 program. The raw reads in FASTQ format were processed, and then the high-quality clean reads were obtained after removing adaptor, ploy-N and low-quality sequences. The final clean reads were assembled using Trinity software (version 2). For annotation, all assembled unigenes were searched against a number of public databases, including the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), the non-redundant nucleotide sequences (Nt), the Protein Family (Pfam), the Clusters of Orthologous Groups of protein database (COG), the Swiss-Prot protein database, the GO and the KEGG databases. The expression level of each gene was analyzed and represented by the expected number of fragments per kilobase of transcript sequence per millions base pairs (FPKM) using RSEM software with default settings [74]. Differentially expressed genes between two P treatments were identified using DESeq2 [75]. Genes with q-value <0.05 and |log2(fold change)| ≥1 were assigned as differentially expressed. GO and KEGG enrichment analyses of DEGs were performed as previously described [71, 76]. The interaction networks were analyzed by Cytoscape (version 3.8.0). The raw data were deposited in the Gene Expression Omnibus under GEO series number GSE171448. A total of 13 DEGs were selected to assess the accuracy of RNA-seq data using qRT-PCR method. qRT-PCR analysis was performed according to SYBR Green Master Mix kit (Vazyme, China), and was monitored on a QuantStudio™ 6 Flex Real-Time System (Thermo Fisher Scientific, Waltham, MA, USA). Specific primers of the tested genes are listed in Additional file 12. The relative expression of candidate gene was calculated relative to the expression of reference gene SgEF-1a as previously described [37]. Three biological replicates were included in the qRT-PCR analysis. Data analysis was performed for the mean and standard error calculation using Microsoft Excel 2003 (Microsoft Company, USA). One-way ANOVA and Student’s t-test analyses were performed with the SPSS program (SPSS Institute, USA, v. 13.0).Methods
Plant growth and treatments
Determination of root morphology and P concentration
Analysis of APase activity
RNA extraction and sequencing
Transcriptomic analysis
Validation of DEGs by qRT-PCR analysis
Statistical analysis
Availability of data and materials
The datasets are included in this article and its Additional files are available from the corresponding author on reasonable request. The datasets for this study can be found in the NCBI Gene Expression Omnibus under GEO series number of GSE171448.
Abbreviations
- ACP:
-
Acid phosphatase
- ALMT:
-
Aluminum-activated malate transporter
- ATP:
-
Adenosine triphosphate
- BP:
-
Biological processes
- CC:
-
Cellular components
- COG:
-
Clusters of orthologous groups of protein database
- DEGs:
-
Differentially expressed genes
- dNTP:
-
Deoxynucleoside triphosphate
- FPKM:
-
Expected number of fragments per kilobase of transcript sequence per millions base pairs
- GO:
-
Gene ontology
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- MATE:
-
Multidrug and toxin extrusion
- MF:
-
Molecular function
- NCBI:
-
National center for biotechnology information
- Nr:
-
Non-redundant protein sequences
- Nt:
-
Non-redundant nucleotide sequences
- PAP:
-
Purple acid phosphatase
- Pfam:
-
Protein family
- PHR1:
-
PHOSPHATE STARVATION RESPONSE 1
- PPCK:
-
Phosphoenolpyruvate carboxylase kinase
- PSI:
-
Pi starvation induced
- PSR:
-
Pi starvation responses
- PT:
-
Pi transporter
- qRT-PCR:
-
Quantitative real-time polymerase chain reaction
- RNA-seq:
-
RNA sequencing
- RNS:
-
Ribonuclease
- SPX:
-
SYG1/PHO81/XPR1
- TFs:
-
Transcription factors
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Acknowledgements
We thank **aoyan Zou for assistance in preparation of figures and Chun Liu for providing critical comments about this work.
Funding
The research was supported by the National Natural Science Foundation of China (31801951, 31861143013, 31802125), the Modern Agro-industry Technology Research System (CARS-34), and the Integrated Demonstration of Key Techniques for the Industrial Development of Featured Crops in Rocky Desertification Areas of Yunnan-Guangxi-Guizhou Provinces (SMH2019-2021). The authors declare that none of the funding bodies have any role in the design of the study or collection, analysis, and interpretation of data as well as in writing the manuscript.
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Contributions
X.M. and G.L. designed the research. Z.C., J.S. and X.L. performed the physiological experiments. Z.C. J.A. and J.A.C. carried out the transcriptomic analysis. Z.C., X.M., G.L., I.R., R.S.K., M.P. and X.M. analyzed the data and wrote the article. All authors read and approved the final manuscript.
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The stylo seeds used in this study were provided by the Tropical Pasture Research Center, Institute of Tropical Crop Genetic Resources (TCGRI), Chinese Academy of Tropical Agriculture Sciences (CATAS), Hainan, China. All experimental researches were performed in accordance with the institutional and national guidelines of China.
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Supplementary Information
Additional file 1: Table S1.
The decrease rate of growth parameters of stylo under 0 (LP) and 100 μM KH2PO4 relative to that in 250 (HP) μM KH2PO4 treatment.
Additional file 2: Table S2.
Summary of stylo roots transcriptomes in 0 (LP) and 250 (HP) μM KH2PO4 supply treatments.
Additional file 3: Table S3.
DEGs identified in stylo roots under 0 (LP) and 250 (HP) μM KH2PO4 supply treatments.
Additional file 4: Figure S1.
Clustering analysis of the DEGs under two P conditions. The transcripts of DEGs were normalized as log2(FPKM). Expression levels ranged from red to blue indicate high to low expression for genes, respectively. LP and HP represent 0 and 250 μM KH2PO4 supply treatments, respectively.
Additional file 5: Table S4.
DEGs related to transporters.
Additional file 6: Table S5.
DEGs encoding putative phosphatases.
Additional file 7: Table S6.
DEGs related to root growth.
Additional file 8: Table S7.
Potential transcription factors involved in Pi signaling.
Additional file 9: Table S8
. DEGs related to plant hormone signal transduction.
Additional file 10: Figure S2.
Heatmap analysis of the DEGs belonging to SPX containing proteins. The transcripts of DEGs were normalized as log2(FPKM+1). Gene IDs were showed by the legend on the right. Expression levels ranged from red to blue indicate high to low expression for genes, respectively. LP and HP represent 0 and 250 μM KH2PO4 supply treatments, respectively.
Additional file 11: Figure S3.
Correlation analysis of gene expression between transcriptome data and qRT-PCR results. Nine up-regulated and four down-regulated DEGs were selected for qRT-PCR analysis. Transcriptome data were plotted against data from qRT-PCR. Data are presented on a log2 scale.
Additional file 12: Table S9.
Primers used for qRT-PCR analysis of the selected genes.
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Chen, Z., Song, J., Li, X. et al. Physiological responses and transcriptomic changes reveal the mechanisms underlying adaptation of Stylosanthes guianensis to phosphorus deficiency. BMC Plant Biol 21, 466 (2021). https://doi.org/10.1186/s12870-021-03249-2
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DOI: https://doi.org/10.1186/s12870-021-03249-2