Identification and expression analysis of phosphate transporter genes and metabolites in response to phosphate stress in Capsicum annuum
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
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These authors contributed equally to this work.