Identification and expression analysis of phosphate transporter genes and metabolites in response to phosphate stress in Capsicum annuum

https://doi.org/10.1016/j.envexpbot.2021.104597Get rights and content

Highlights

  • A total of twenty-eight phosphate transporter genes (CaPHT and CaPHO) were identified in Capsicum annuum.

  • C. annuum PHTs showed a high syntenic and evolutionary relationship with tomato and potato.

  • CaPHT and CaPHO genes showed high 3D protein structural similarity with PHT reference models.

  • Expression of most of the CaPHT1 and CaPHO genes were induced during P-stress.

  • Most of the sugar metabolites showed high variations in both leaf and root tissues under P-stress.

  • Under low P-stress, majority of the organic acids content decreased in leaf tissues.

Abstract

Till date, no systematic study of phosphate transporter (PHT) genes is reported in Capsicum species. For the first time, we report the identification and homology modelling of C. annuum PHT genes (CaPHT) belonging to CaPHT1, CaPHT2, CaPHT3, CaPHT4, CaPHT5 and CaPHO family. Further, gene duplication analysis identified 14 segmental and 5 tandemly duplicated gene pairs. Synteny analysis showed high collinearity of CaPHTs with tomato and potato. The 3D structures of CaPHT genes were highly similar to the reference model suggesting potential functional similarities of PHT genes in Capsicum. Analysis in fruit transcriptomes and validation using quantitative real-time PCR showed differential expressions (DE) of CaPHTs in leaf, stem, root, flower and fruit tissues of C. annuum. During P-stress conditions CaPHT1;3 to CaPHT1;7, CaPHT3;1 to CaPHT3;4 and six members of CaPHO family showed DE patterns between leaf and root tissues. Metabolite profiling of plants grown under P-stress conditions showed significantly (p-value <0.001) higher content of glucose, fructose and erythritol in leaves compared to roots. Furthermore, it was observed that most of the organic acids content in leaf decreased under P-stress. The identified CaPHTs would serve as a resource for further studies of phosphate uptake and homeostasis in Capsicum.

Introduction

For normal growth and development, plants required one of the essential macroelements known as phosphorus (P), which is an integral part of several biological processes, including metabolic reactions, photosynthesis, signal transduction, genetic transfer, nutrient transport (Blevins, 1999; Hawkesford et al., 2012). In soil, P occurs mainly in two forms, i.e., organic and inorganic phosphate (Pi); however, the latter one, i.e. Pi is acquired by plants primarily in the form of orthophosphate (H2PO4 and H2PO42−; (Richardson et al., 2009; Shen et al., 2011; Vance et al., 2003))and is absorbed directly by roots (Lambers and Plaxton, 2018). Despite the copious amount of P, the availability of Pi is usually limited to plants for direct use due to the poor diffusion rate and its high affinity with other elements such as calcium (Ca), iron (Fe), and aluminium (Al) in the soil (Oelkers and Valsami-Jones, 2008; Pierzynski et al., 2005; Smith et al., 2003). Therefore, to overcome the scarcity of available Pi and to boost Pi uptake, plants have acquired several approaches to increase the Pi absorption, for instance, plants adapted via changing the root morphology (Müller and Schmidt, 2004; Savage et al., 2013; Williamson et al., 2001) and which increases the affinity of Pi transporters in root hair (Mudge et al., 2002) to absorb Pi. Moreover, in plants, the transporters which facilitate the Pi transportation are categorized into two, i.e. high and low-affinity phosphate transporters (PHTs; (Misson et al., 2004; Poirier and Bucher, 2002; Raghothama, 2000)). At natural Pi soil condition or a low Pi concentration (μM scale), the high-affinity actively maintain the plant Pi requirement (Poirier and Bucher, 2002), while at an optimum Pi concentration (mM scale), the low-affinity PHTs actively operate for Pi supply (Karthikeyan et al., 2002; Raghothama, 2000). To maintain the Pi homeostasis in plants, several genes are consecutively expressed under high and low Pi situations (Raghothama, 2000; Yang et al., 2020). Based on sequence similarity, these Pi transporter genes are catalogued into six different PHT families, from PHT1 to PHT5 and PHO, which are intrinsically or transcriptionally expressed (Liu et al., 2016; Młodzińska and Zboińska, 2016; Wang et al., 2017). After the discovery of high-affinity PHT gene PHO84 in yeast (Bun-Ya et al., 1991), genes or the members of the different PHTs families have been identified in several plant species, including A. thaliana (Guo et al., 2008; Muchhal et al., 1996; Okumura et al., 1998), wheat (Teng et al., 2017), rice (Dong et al., 2019; Liu et al., 2011; Ruili et al., 2020), tomato (Chen et al., 2014), potato (Cao et al., 2020; Liu et al., 2017), Poplar (Zhang et al., 2016), apple (Sun et al., 2017), sorghum (Wang et al., 2019) and maize (Liu et al., 2018). Among the PHT families, the PHT1 is the most studied family in plants which contains 12 transmembrane domains (TMDs) and facilitate Pi acquisition from soil navigated by plasma membrane H+-ATPase (Ullrich-Eberius et al., 1981). The PHT2, a low-affinity Pi transporter with 12 TMDs, is found in the inner envelope membrane of the chloroplast and is responsible for transferring Pi inside the leaves (Daram et al., 1999; Shi et al., 2013; Versaw and Harrison, 2002). Members of the PHT2 family are expressed in green tissues and roots (Guo et al., 2013; Zhang et al., 2016). The PHT3 family, also known as mitochondrial PHT (mPHT), is a high-affinity Pi transporter and plays a crucial role in Pi transport between the cytoplasm and mitochondrial matrix (Zhu et al., 2012). The PHT3 members were also reported to be involved in plant development and maintain Pi homeostasis essential for plant stress tolerance (Shukla et al., 2016; Takabatake et al., 1999; Zhang et al., 2016). Further, PHT4 family members, structurally similar to animal SLC17/type I transporters, were reported to be involved in the Pi transportation between plastids, Golgi apparatus and between cytosol and chloroplast (Ferro et al., 2003; Guo et al., 2008; Roth et al., 2004; Ruili et al., 2020). The PHT4 members are mainly expressed in root and leave tissues (Guo et al., 2008) and are found to be involved in protein N-glycosylation, cell wall biosynthesis, salt tolerance(Cubero et al., 2009; Ruili et al., 2020) and in photostress (Miyaji et al., 2015). Furthermore, PHT5 members or vacuolar Pi transporters (VPTs) were identified to transport Pi across the vacuole to regulate Pi homeostasis in the cytoplasm (Liu et al., 2015, 2016). The PHT5 family is also important in the adaptation of plants with varying Pi levels in the environment and is essential for better plant growth and fitness (Liu et al., 2015, 2016). Like PHT1, members of the PHO family also facilitate the Pi acquisition from soil and transport it to xylem and other plant tissues (Młodzińska and Zboińska, 2016; Wang et al., 2004). At low Pi or Pi starvation, PHO1 provide root to shoot signal transduction cascade (Hamburger et al., 2002; Stefanovic et al., 2007) and act as Pi exporter associated with protein localization in Golgi and trans-Golgi networks (Arpat et al., 2012; Wege et al., 2016). In addition, plants showed adaptation of Pi regulation to maximize Pi acquisition via modification of several metabolic processes sporadically coordinated by expression of Pi deficiency responsive genes (Morcuende et al., 2007; Wasaki et al., 2003; Wu et al., 2003). Also, in plants, changes in several metabolic processes, including organic acid synthesis, metabolism of carbohydrates, ammonium metabolism and lipids were observed during phosphate starvation (Huang et al., 2008; Morcuende et al., 2007; Wasaki et al., 2003).

Capsicum, aka chili is a diploid (2n = 24), a self-pollinating crop from the Solanaceae family, diverged ∼19.6 million years ago (MYA) from tomato and potato (Kim et al., 2017, 2014). It is an economically important vegetable crop grown worldwide and used as a spice and colouring agent and in pharmaceutical industries. The five cultivated species of chili peppers are C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens (Aguilar‐Meléndez et al., 2009). In Capsicum, it was reported that the usage of fertilizer in irrigation water affects the phosphorus mobilization or Pi uptake (Silber et al., 2005). Another study reported decreased leaf Pi and total phosphorus levels in C. annuum leaves gradually developed within 3–4 days of mild degree of water stress (Turner, 1985). However, a comprehensive study of phosphate transporter (PHT) genes in C. annuum has not been reported yet. Therefore, in this study, we report identification of PHT genes in C. annuum and their expression analysis in normal and phosphate stress (P-stress) induced plants for the first time. We also investigated the changes in metabolites expression/content in response to phosphate stress conditions. The results of this study consequently may provide a basic foundation for further studies of PHT genes in Capsicum species.

Section snippets

Identification of phosphate transporter genes in C. annuum genome

For the identification of phosphate transporter (CaPHT) gene family members, the proteome, CDS sequences and the genome information of C. annuum (Zunla 1; (GCF_000710875.1; (Kim et al., 2017)) were downloaded from NCBI (www.ncbi.nlm.nih.gov). The protein sequences of Arabidopsis thaliana (A. thaliana) PHT (AtPHT) gene family members were downloaded from the Arabidopsis Information Resource (TAIR; release 10; (Lamesch et al., 2012)) database. Subsequently, C. annuum protein sequences were

Identification and phylogenetic classification of CaPHT gene family

A total of 28 putative CaPHTs genes (8 CaPHT1, 1 CaPHT2, 4 CaPHT3, 6 CaPHT4, 3 CaPHT5 and 6 CaPHO) were identified from the C. annuum genome. Their basic description, including genomic locations, protein and CDS length, exon counts, Mw, theoretical isoelectric point (pI), subcellular localization, hydrophobic index and TMDs information were listed in Table S2. The length of 28 CaPHT proteins ranged from 313 to 798 amino acids, while their molecular weight (Mw) ranged between 34.62 to 92.45 kDa

Discussion

Phosphorus is a key constituent of many molecules (ATP, nucleic acids, and phospholipids) and involved in the regulation of several biological processes, including cell signalling, energy transfer and stress tolerance (Raghothama, 2000; Wang et al., 2017). In plants, several genes are responsible for the acquisition and transportation of phosphate and are known as phosphate transporters (PHTs). To date, multiple PHT families have been identified in a wide number of plants (Cao et al., 2020;

Conclusion

This study is the first thorough investigation of C. annuum phosphate transporters (CaPHT) genes. The present study identified 28 CaPHTs genes which were classified into six groups (CaPHT1 to CaPHT5 and CaPHO) based on their conserved motifs and phylogenetic relationships to Arabidopsis. Of 28 CaPHTs, 14 segmental and 5 tandemly duplicated gene pairs indicate their purifying selection during evolution. Further, CaPHTs genes showed higher collinearity with tomato and potato of the same

Author statement

Ilyas Ahmad: Methodology, Formal analysis, Investigation, Data Curation, Visualization, Writing, Original draft preparation. Abdul Rawoof: Methodology, Formal analysis, Investigation, Data Curation, Visualization, Writing, Original draft preparation. Khushbu Islam: Formal analysis, Writing, Reviewing and Editing. John Momo: Formal analysis, Writing, Reviewing and Editing. Nirala Ramchiary: Conceptualization, Methodology, Writing, Original draft preparation, Resources, Supervision, Project

Author’s contributions

NR conceived and designed the experiment. IA, AR, KI and JM performed the experiments. IA and AR analyzed the data. IA and AR drafted and wrote the manuscript. NR corrected and finalized the manuscript. All authors contributed in drafting and revision and approved the final manuscript.

Availability of transcriptome data

The RNA sequencing data of fruit samples (EG and Br stage) of C. annuum used in this study was submitted to NCBI under bioproject (PRJNA679780). Sequence Read Archive (SRA) accessions SRR12963501, SRR12963503 and SRR12963504 are EG fruit samples and SRR12963498, SRR12963499, SRR12963500 are of Breaker fruit samples.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This work was supported by the Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India in the form of Ramalingaswami Re-entry Fellowship cum Research Grant. The authors acknowledge the funding received from the Department of Science and Technology (DST), Govt. of India, in the form of DST FIST-II given to School of Life Sciences, Jawaharlal Nehru University, New Delhi. IA and JM acknowledge Senior Research fellowships (SRF) received from the Council of Scientific

References (110)

  • L.B. Turner

    Changes in the phosphorus content of Capsicum annuum leaves during water stress

    J. Plant Physiol.

    (1985)
  • D. Wang et al.

    KaKs_calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies

    Genomics Proteomics Bioinformatics

    (2010)
  • A. Aguilar‐Meléndez et al.

    Genetic diversity and structure in semiwild and domesticated chiles (Capsicum annuum; Solanaceae) from Mexico

    Am. J. Bot.

    (2009)
  • A.B. Arpat et al.

    Functional expression of PHO1 to the Golgi and trans‐Golgi network and its role in export of inorganic phosphate

    Plant J.

    (2012)
  • T.L. Bailey et al.

    MEME SUITE: tools for motif discovery and searching

    Nucleic Acids Res.

    (2009)
  • D.G. Blevins

    Why plants need phosphorus

    Better Crop.

    (1999)
  • M. Bucher

    Functional biology of plant phosphate uptake at root and mycorrhiza interfaces

    New Phytol.

    (2007)
  • M. Bun-Ya et al.

    The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter

    Mol. Cell. Biol.

    (1991)
  • M. Cao et al.

    Functional Analysis of StPHT1; 7, a Solanum tuberosum L. Phosphate Transporter Gene, in Growth and Drought Tolerance

    Plants

    (2020)
  • S.A. Ceasar

    Genome‐wide Identification and in silico analysis of PHT1 family genes and proteins in Setaria viridis: the best model to study nutrient transport in millets

    Plant Genome

    (2019)
  • A. Chen et al.

    Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species

    New Phytol.

    (2007)
  • A. Chen et al.

    Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of Pht1 family genes in response to Pi deficiency in tomato

    BMC Plant Biol.

    (2014)
  • C. Choi et al.

    Molecular characterization of Oryza sativa WRKY 6, which binds to W‐box‐like element 1 of the Oryza sativa pathogenesis‐related (PR) 10a promoter and confers reduced susceptibility to pathogens

    New Phytol.

    (2015)
  • P. Daram et al.

    Pht2; 1 encodes a low-affinity phosphate transporter from Arabidopsis

    Plant Cell

    (1999)
  • Z. Dong et al.

    The rice phosphate transporter protein OsPT8 regulates disease resistance and plant growth

    Sci. Rep.

    (2019)
  • S. El-Gebali et al.

    The Pfam protein families database in 2019

    Nucleic Acids Res.

    (2019)
  • A.H. Ganie et al.

    Metabolite profiling of low-P tolerant and low-P sensitive maize genotypes under phosphorus starvation and restoration conditions

    PLoS One

    (2015)
  • P. García-Caparrós et al.

    Phosphorus and carbohydrate metabolism in green bean plants subjected to increasing phosphorus concentration in the nutrient solution

    Agronomy

    (2021)
  • E. Gasteiger et al.

    Protein identification and analysis tools on the ExPASy server

    The Proteomics Protocols Handbook

    (2005)
  • Z. Gu et al.

    Circlize implements and enhances circular visualization in R

    Bioinformatics

    (2014)
  • B. Guo et al.

    Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters

    New Phytol.

    (2008)
  • C. Guo et al.

    Function of wheat phosphate transporter gene TaPHT2; 1 in Pi translocation and plant growth regulation under replete and limited Pi supply conditions

    Planta

    (2013)
  • D. Hamburger et al.

    Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem

    Plant Cell

    (2002)
  • J.P. Hammond et al.

    Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

    Plant Physiol.

    (2003)
  • M.J. Harrison et al.

    A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

    Plant Cell

    (2002)
  • P. Horton et al.

    WoLF PSORT: protein localization predictor

    Nucleic Acids Res.

    (2007)
  • C.Y. Huang et al.

    Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in phosphate-deficient barley plants (Hordeum vulgare L.)

    Plant Cell Physiol.

    (2008)
  • S. Islam et al.

    Genome-wide identification of glutathione S-transferase gene family in pepper, its classification, and expression profiling under different anatomical and environmental conditions

    Sci. Rep.

    (2019)
  • L.S. Johnson et al.

    Hidden Markov model speed heuristic and iterative HMM search procedure

    BMC Bioinformatics

    (2010)
  • A.S. Karthikeyan et al.

    Regulated expression of Arabidopsis phosphate transporters

    Plant Physiol.

    (2002)
  • A. Kaur et al.

    In-silico analysis of cis-acting regulatory elements of pathogenesis-related proteins of Arabidopsis thaliana and Oryza sativa

    PLoS One

    (2017)
  • L.A. Kelley et al.

    The Phyre2 web portal for protein modeling, prediction and analysis

    Nat. Protoc.

    (2015)
  • S. Kim et al.

    Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species

    Nat. Genet.

    (2014)
  • S. Kim et al.

    New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication

    Genome Biol.

    (2017)
  • D. Kim et al.

    Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype

    Nat. Biotechnol.

    (2019)
  • S. Kumar et al.

    MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets

    Mol. Biol. Evol.

    (2016)
  • H. Lambers et al.

    Phosphorus: back to the roots

    Annu. Plant Rev. Online

    (2018)
  • P. Lamesch et al.

    The Arabidopsis Information Resource ({TAIR}): improved gene annotation and new tools

    Nucleic Acids Res.

    (2012)
  • M. Lescot et al.

    PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences

    Nucleic Acids Res.

    (2002)
  • I. Letunic et al.

    20 years of the SMART protein domain annotation resource

    Nucleic Acids Res.

    (2018)
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