A genetically validated approach to detect inorganic polyphosphates in plants

Abstract

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. PolyPs represent important stores of phosphate and energy, and are abundantly found in many pro-and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabiopsis cells by heterologous expression of E. coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme lead to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1 expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron dense structures in transmission electron microscopy (TEM) sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono-and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalicisomes in the green algae Chlamydomonas reinhardtii. Together, our experiments suggest that higher plants may not contain large polyPs stores. Keywordsinorganic polyphosphate, energy metabolism, polyphosphate kinase, polyphosphate phosphatase, Arabidopsis, Chlamydomonas Significance StatementA chemical dye and an inorganic polyphosphate binding domain are shown to specifically label inorganic polyphosphate granules in transgenic Arabidopsis lines and Chlamydomonas acidocalcisomes. Using these tools, we show that in contrast to many prokaryotic and eukaryotic organisms, higher plants do not seem to contain large inorganic polyphosphate stores.

Fixing the Broken Phosphorus Cycle: Wastewater Remediation by Microalgal Polyphosphates

“…In place of PPK, a VTC complex located at the vacuole or acidocalcisomes appears to be responsible for PolyP synthesis in yeast (Hothorn et al, 2009) and Chlamydomonas (Aksoy et al, 2014). In higher plants the existence of the VTC complex is uncertain, and the presence of large stores of PolyP has been questioned (Zhu et al, 2020). Figure 3 shows a schematic of poly P synthesis based on the yeast model (with putative or known Chlamydomonas orthologues indicated).…”

“…These tend to accumulate in vacuoles or acidocalcisomes as granules ( Albi and Serrano, 2016 ). Although higher plants produce organic phosphate (P o ) in the form of phytate (inositol hexakisphosphate) there is no strong evidence to support the presence of actual PolyP granules in higher plants, despite a recent and extensive search ( Zhu et al, 2020 ). Instead, these can be found in bacteria, fungi, and lower plants, such as algae and the mosses ( Seufferheld and Curzi, 2010 ).…”

“…PolyP-DAPI can be used for measurement in plate assays but there is much spectral overlap with DNA-DAPI fluorescence ( Shebanova et al, 2017 ). A PolyP-specific dye has been identified for both microscopy and quantitative plate assays ( Angelova et al, 2014 ) but has only recently been exploited ( Zhu et al, 2020 ) 1 . PolyP can also be separated by chain length by electrophoresis in DNA-sequencing gels or commercial mini-gels ( Smith et al, 2018 ).…”

“…The occurrence of these genes is very limited in eukaryotes however ( Rao et al, 2009 ) and no orthologues of these genes could be detected in the Chlamydomonas genome. Over-expression of E.coli Ppk1 in yeast ( Gerasimaite et al, 2014 ) and Arabidopsis ( Zhu et al, 2020 ) has led to toxicity (possibly due to cytosolic PolyP accumulation) and in the latter case, ectopic PolyP granules were identified.…”

“…Endopolyphosphatases (PPN) have also been identified in yeast ( Albi and Serrano, 2016 ). The enzymes involved in the mobilization of PolyP have yet to be fully studied in microalgae, although various polyphosphatases have been identified in higher plants ( Zhu et al, 2020 ). Understanding these factors will help target genes for increasing PolyP levels in microalgae for P-remediation.…”