PHOSPHATE1-mediated phosphate translocation from roots to shoots regulates floral transition in plants.

Phosphorus nutrition has been known to influence floral transition in plants for a long time, but the underlying mechanism is unclear. Arabidopsis PHOSPHATE1 (PHO1) plays a critical role in phosphate translocation from roots to shoots, but whether and how it regulates floral transition is unknown. Here, we show that knockout mutation of PHO1 delays flowering under both long-day and short-day conditions. The late flowering of pho1 mutants can be partially rescued by Pi supplementation in rosettes or shoot apices. Grafting assay indicates that the late flowering of pho1 mutants is resulted from impaired phosphate translocation from roots to shoots. Knockout mutation of SPX1 and SPX2, two negative regulators of phosphate starvation response, partially rescues the late flowering of pho1 mutants. PHO1 is epistatic to PHO2, a negative regulator of PHO1, in flowering time regulation. Loss of PHO1 represses the expression of some floral activators, including FT encoding florigen, and induces the expression of some floral repressors in shoots. Genetic analyses indicate that at least jasmonic acid signaling is partially responsible for the late flowering of pho1 mutants. In addition, we find rice PHO1;2, the homology of PHO1, plays a similar role in floral transition. These results suggest that PHO1 integrates phosphorus nutrition and flowering time and could be used as a potential target in modulating phosphorus nutrition-mediated flowering time in plants.

Calmodulin and calmodulin-like protein-mediated plant responses to biotic stresses.

Plants have evolved a set of finely regulated mechanisms to respond to various biotic stresses. Transient changes in intracellular calcium (Ca2+ ) concentration have been well documented to act as cellular signals in coupling environmental stimuli to appropriate physiological responses with astonishing accuracy and specificity in plants. Calmodulins (CaMs) and calmodulin-like proteins (CMLs) are extensively characterized as important classes of Ca2+ sensors. The spatial-temporal coordination between Ca2+ transients, CaMs/CMLs and their target proteins is critical for plant responses to environmental stresses. Ca2+ -loaded CaMs/CMLs interact with and regulate a broad spectrum of target proteins, such as ion transporters (including channels, pumps, and antiporters), transcription factors, protein kinases, protein phosphatases, metabolic enzymes and proteins with unknown biological functions. This review focuses on mechanisms underlying how CaMs/CMLs are involved in the regulation of plant responses to diverse biotic stresses including pathogen infections and herbivore attacks. Recent discoveries of crucial functions of CaMs/CMLs and their target proteins in biotic stress resistance revealed through physiological, molecular, biochemical, and genetic analyses have been described, and intriguing insights into the CaM/CML-mediated regulatory network are proposed. Perspectives for future directions in understanding CaM/CML-mediated signalling pathways in plant responses to biotic stresses are discussed. The application of accumulated knowledge of CaM/CML-mediated signalling in biotic stress responses into crop cultivation would improve crop resistance to various biotic stresses and safeguard our food production in the future.

Sucrose rather than GA transported by AtSWEET13 and AtSWEET14 supports pollen fitness at late anther development stages.

Both sugar and the hormone gibberellin (GA) are essential for anther-enclosed pollen development and thus for plant productivity in flowering plants. Arabidopsis (Arabidopsis thaliana) AtSWEET13 and AtSWEET14, which are expressed in anthers and associated with seed yield, transport both sucrose and GA. However, it is still unclear which substrate transported by them directly affects anther development and seed yield. Histochemical staining, cross-sectioning and microscopy imaging techniques were used to investigate and interpret the phenotypes of the atsweet13;14 double mutant during anther development. Genetic complementation of atsweet13;14 using AtSWEET9, which transports sucrose but not GA, and the GA transporter AtNPF3.1, respectively, was conducted to test the substrate preference relevant to the biological process. The loss of both AtSWEET13 and AtSWEET14 resulted in reduced pollen viability and therefore decreased pollen germination. AtSWEET9 fully rescued the defects in pollen viability and germination of atsweet13;14, whereas AtNPF3.1 failed to do so, indicating that AtSWEET13/14-mediated sucrose rather than GA is essential for pollen fertility. AtSWEET13 and AtSWEET14 function mainly at the anther wall during late anther development stages, and they probably are responsible for sucrose efflux into locules to support pollen development to maturation, which is vital for subsequent pollen viability and germination.

Overexpression of a Plasma Membrane H+-ATPase Gene OSA1 Stimulates the Uptake of Primary Macronutrients in Rice Roots.

Plasma membrane (PM) H+-ATPase is a master enzyme involved in various plant physiological processes, such as stomatal movements in leaves and nutrient uptake and transport in roots. Overexpression of Oryza sativa PM H+-ATPase 1 (OSA1) has been known to increase NH4+ uptake in rice roots. Although electrophysiological and pharmacological experiments have shown that the transport of many substances is dependent on the proton motive force provided by PM H+-ATPase, the exact role of PM H+-ATPase on the uptake of nutrients in plant roots, especially for the primary macronutrients N, P, and K, is still largely unknown. Here, we used OSA1 overexpression lines (OSA1-oxs) and gene-knockout osa1 mutants to investigate the effect of modulation of PM H+-ATPase on the absorption of N, P, and K nutrients through the use of a nutrient-exhaustive method and noninvasive microtest technology (NMT) in rice roots. Our results showed that under different concentrations of P and K, the uptake rates of P and K were enhanced in OSA1-oxs; by contrast, the uptake rates of P and K were significantly reduced in roots of osa1 mutants when compared with wild-type. In addition, the net influx rates of NH4+ and K+, as well as the efflux rate of H+, were enhanced in OSA1-oxs and suppressed in osa1 mutants under low concentration conditions. In summary, this study indicated that overexpression of OSA1 stimulated the uptake rate of N, P, and K and promoted flux rates of cations (i.e., H+, NH4+, and K+) in rice roots. These results may provide a novel insight into improving the coordinated utilization of macronutrients in crop plants.

Insights of intracellular/intercellular phosphate transport and signaling in unicellular green algae and multicellular land plants.

Phosphorus (P) is an essential element for plant growth and development. Vacuoles play a fundamental role in the storage and remobilization of P in plants, while our understanding of the evolutionary mechanisms of creating and reusing P stores are limited. Besides, we also know very little about the coordination of intercellular P translocation, neither the inorganic phosphate (Pi) signaling nor the Pi transport patterns. Here we summarize recent advances in understanding the core elements involved in cellular and/or subcellular P homeostasis and signaling in unicellular green algae and multicellular land plants. We also propose further work that might help to uncover the high-resolution intracellular and intercellular landscape of Pi distribution and signaling in plants.

Combined analyses of translatome and transcriptome in Arabidopsis reveal new players responding to magnesium deficiency.

Translational control of gene expression, including recruitment of ribosomes to messenger RNA (mRNA), is particularly important during the response to stress. Purification of ribosome-associated mRNAs using translating ribosome affinity purification (TRAP) followed by RNA-sequencing facilitates the study of mRNAs undergoing active transcription and better proxies the translatome, or protein response, to stimuli. To identify plant responses to Magnesium (Mg) deficiency at the translational level, we combined transcriptome and translatome analyses. Excitingly, we found 26 previously unreported Mg-responsive genes that were only regulated at the translational level and not the transcriptional level, during the early response to Mg deficiency. In addition, mutants of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), the H+ /CATION EXCHANGER 1 and 3 (CAX1 and CAX3), and UBIQUITIN 11 (UBQ11) exhibited early chlorosis phenotype under Mg deficiency, supporting their functional involvement in ion homeostasis. Overall, our study strongly supports that TRAP-seq combined with RNA-seq followed by phenotype screening could facilitate the identification of novel players during stress responses.

Integrated analyses of miRNAome and transcriptome reveal zinc deficiency responses in rice seedlings.

BACKGROUND: Zinc (Zn) deficiency is one of the most widespread soil constraints affecting rice productivity, but the molecular mechanisms underlying the regulation of Zn deficiency response is still limited. Here, we aim to understand the molecular mechanisms of Zn deficiency response by integrating the analyses of the global miRNA and mRNA expression profiles under Zn deficiency and resupply in rice seedlings by integrating Illumina's high-throughput small RNA sequencing and transcriptome sequencing. RESULTS: The transcriptome sequencing identified 360 genes that were differentially expressed in the shoots and roots of Zn-deficient rice seedlings, and 97 of them were recovered after Zn resupply. A total of 68 miRNAs were identified to be differentially expressed under Zn deficiency and/or Zn resupply. The integrated analyses of miRNAome and transcriptome data showed that 12 differentially expressed genes are the potential target genes of 10 Zn-responsive miRNAs such as miR171g-5p, miR397b-5p, miR398a-5p and miR528-5p. Some miRNA genes and differentially expressed genes were selected for validation by quantitative RT-PCR, and their expressions were similar to that of the sequencing results. CONCLUSION: These results provide insights into miRNA-mediated regulatory pathways in Zn deficiency response, and provide candidate genes for genetic improvement of Zn deficiency tolerance in rice.

Transcriptome profiles of soybean leaves and roots in response to zinc deficiency.

Zinc (Zn) deficiency is a widespread agricultural problem in arable soils of the whole world. However, the molecular mechanisms underlying Zn-deficiency response are largely unknown. Here, we analyzed the transcriptomic profilings of soybean leaves and roots in response to Zn deficiency through Illumina's high-throughput RNA sequencing in order to understand the molecular basis of Zn-deficiency response in the plants. A total of 614 and 1011 gene loci were found to be differentially expressed in leaves and roots, respectively, and 88 loci were commonly found in both leaves and roots. Twelve differentially expressed genes (DEGs) were randomly selected for validation by quantitative reverse transcription polymerase chain reaction, and their fold changes were similar to those of RNA-seq. Gene ontology enrichment analysis showed that ion transport, nicotianamine (NA) biosynthetic process and queuosine biosynthetic process were enriched in the upregulated genes, while oxidation-reduction process and defense response were enriched in the downregulated genes. Among the DEGs, 20 DEGs are potentially involved in Zn homeostasis, including seven ZRT, IRT-related protein (ZIP) transporter genes, three NA synthase genes, and seven metallothionein genes; 40 DEGs are possibly involved in diverse hormonal signals such as auxin, cytokinin, ethylene and gibberellin; nine DEGs are putatively involved in calcium signaling; 85 DEGs are putative transcription factor genes. Nine DEGs were found to contain zinc-deficiency-response element in their promoter regions. These results could provide comprehensive insights into the soybean response to Zn deficiency and will be helpful for further elucidation of the molecular mechanisms of Zn-deficiency response and Zn-deficiency tolerance in plants.

Early Transcriptomic Response to Phosphate Deprivation in Soybean Leaves as Revealed by RNA-Sequencing.

Low phosphate (Pi) availability is an important limiting factor affecting soybean production. However, the underlying molecular mechanisms responsible for low Pi stress response and tolerance remain largely unknown, especially for the early signaling events under low Pi stress. Here, a genome-wide transcriptomic analysis in soybean leaves treated with a short-term Pi-deprivation (24 h) was performed through high-throughput RNA sequencing (RNA-seq) technology. A total of 533 loci were found to be differentially expressed in response to Pi deprivation, including 36 mis-annotated loci and 32 novel loci. Among the differentially expressed genes (DEGs), 303 were induced and 230 were repressed by Pi deprivation. To validate the reliability of the RNA-seq data, 18 DEGs were randomly selected and analyzed by quantitative RT-PCR (reverse transcription polymerase chain reaction), which exhibited similar fold changes with RNA-seq. Enrichment analyses showed that 29 GO (Gene Ontology) terms and 8 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were significantly enriched in the up-regulated DEGs and 25 GO terms and 16 KEGG pathways were significantly enriched in the down-regulated DEGs. Some DEGs potentially involved in Pi sensing and signaling were up-regulated by short-term Pi deprivation, including five SPX-containing genes. Some DEGs possibly associated with water and nutrient uptake, hormonal and calcium signaling, protein phosphorylation and dephosphorylation and cell wall modification were affected at the early stage of Pi deprivation. The cis-elements of PHO (phosphatase) element, PHO-like element and P responsive element were present more frequently in promoter regions of up-regulated DEGs compared to that of randomly-selected genes in the soybean genome. Our transcriptomic data showed an intricate network containing transporters, transcription factors, kinases and phosphatases, hormone and calcium signaling components is involved in plant responses to early Pi deprivation.

Genome-wide identification, expression analysis of GH3 family genes in Medicago truncatula under stress-related hormones and Sinorhizobium meliloti infection.

Auxin plays a pivotal role in the regulation of plant growth and development by controlling the expression of auxin response genes rapidly. As one of the major auxin early response gene families, Gretchen Hagen 3 (GH3) genes are involved in auxin homeostasis by conjugating excess auxins to amino acids. However, how GH3 genes function in environmental stresses and rhizobial infection responses in Medicago truncatula are largely unknown. Here, based on the latest updated M. truncatula genome, a comprehensive identification and expression profiling analysis of MtGH3 genes were performed. Our data showed that most of MtGH3 genes were expressed in tissue-specific manner and were responsive to environmental stress-related hormones. To understand the possible roles of MtGH3 genes involved in symbiosis establishment between M. truncatula and symbiotic bacteria, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expressions of MtGH3 genes during the early phase of Sinorhizobium meliloti infection. The expression levels of most MtGH3 genes were upregulated in shoots and downregulated in roots by S. meliloti infection. The differences in expression responses to S. meliloti infection between roots and shoots were in agreement with the results of free indoleacetic acid (IAA) content measurements. The identification and expression analysis of MtGH3 genes at the early phase of S. meliloti infection may help us to understand the role of GH3-mediated IAA homeostasis in the regulation of nodule formation in model legumes M. truncatula.

Plasma membrane H+-ATPases in mineral nutrition and crop improvement.

Plasma membrane H+-ATPases (PMAs) pump H+ out of the cytoplasm by consuming ATP to generate a membrane potential and proton motive force for the transmembrane transport of nutrients into and out of plant cells. PMAs are involved in nutrient acquisition by regulating root growth, nutrient uptake, and translocation, as well as the establishment of symbiosis with arbuscular mycorrhizas. Under nutrient stresses, PMAs are activated to pump more H+ and promote organic anion excretion, thus improving nutrient availability in the rhizosphere. Herein we review recent progress in the physiological functions and the underlying molecular mechanisms of PMAs in the efficient acquisition and utilization of various nutrients in plants. We also discuss perspectives for the application of PMAs in improving crop production and quality.

Measurement of Total Phosphorus and Polyphosphate in Chlamydomonas reinhardtii.

Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.

Comparative transcriptome analyses under individual and combined nutrient starvations provide insights into N/P/K interactions in rice.

Crops often suffer from simultaneous limitations of multiple nutrients in soils, including nitrogen (N), phosphorus (P) and potassium (K), which are three major macronutrients essential for ensuring growth and yield. Although plant responses to individual N, P, and K deficiency have been well documented, our understanding of the responses to combined nutrient deficiencies and the crosstalk between nutrient starvation responses is still limited. Here, we compared the physiological responses in rice under seven kinds of single and multiple low nutrient stress of N, P and K, and used RNA sequencing approaches to compare their transcriptome changes. A total of 13,000 genes were found to be differentially expressed under all these single and multiple low N/P/K stresses, and 66 and 174 of them were shared by all these stresses in roots and shoots, respectively. Functional enrichment analyses of the DEGs showed that a group of biological and metabolic processes were shared by these low N/P/K stresses. Comparative analyses indicated that DEGs under multiple low nutrient stress was not the simple summation of single nutrient stress. N was found to be the predominant factor affecting the transcriptome under combined nutrient stress. N, P, or K availability exhibited massive influences on the transcriptomic responses to starvation of other nutrients. Many genes involved in nutrient transport, hormone signaling, and transcriptional regulation were commonly responsive to low N/P/K stresses. Some transcription factors were predicted to regulate the expression of genes that are commonly responsive to N, P, and K starvations. These results revealed the interactions between N, P, and K starvation responses, and will be helpful for further elucidation of the molecular mechanisms underlying nutrient interactions.

Genome-wide identification and expression analysis of TCP family genes in Catharanthus roseus.

Introduction: The anti-tumor vindoline and catharanthine alkaloids are naturally existed in Catharanthus roseus (C. roseus), an ornamental plant in many tropical countries. Plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play important roles in various plant developmental processes. However, the roles of C. roseus TCPs (CrTCPs) in terpenoid indole alkaloid (TIA) biosynthesis are largely unknown. Methods: Here, a total of 15 CrTCP genes were identified in the newly updated C. roseus genome and were grouped into three major classes (P-type, C-type and CYC/TB1). Results: Gene structure and protein motif analyses showed that CrTCPs have diverse intron-exon patterns and protein motif distributions. A number of stress responsive cis-elements were identified in promoter regions of CrTCPs. Expression analysis showed that three CrTCP genes (CrTCP2, CrTCP4, and CrTCP7) were expressed specifically in leaves and four CrTCP genes (CrTCP13, CrTCP8, CrTCP6, and CrTCP10) were expressed specifically in flowers. HPLC analysis showed that the contents of three classic TIAs, vindoline, catharanthine and ajmalicine, were significantly increased by ultraviolet-B (UV-B) and methyl jasmonate (MeJA) in leaves. By analyzing the expression patterns under UV-B radiation and MeJA application with qRT-PCR, a number of CrTCP and TIA biosynthesis-related genes were identified to be responsive to UV-B and MeJA treatments. Interestingly, two TCP binding elements (GGNCCCAC and GTGGNCCC) were identified in several TIA biosynthesis-related genes, suggesting that they were potential target genes of CrTCPs. Discussion: These results suggest that CrTCPs are involved in the regulation of the biosynthesis of TIAs, and provide a basis for further functional identification of CrTCPs.

Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves.

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.

H+-ATPases in Plant Growth and Stress Responses.

H+-ATPases, including the phosphorylated intermediate-type (P-type) and vacuolar-type (V-type) H+-ATPases, are important ATP-driven proton pumps that generate membrane potential and provide proton motive force for secondary active transport. P- and V-type H+-ATPases have distinct structures and subcellular localizations and play various roles in growth and stress responses. A P-type H+-ATPase is mainly regulated at the posttranslational level by phosphorylation and dephosphorylation of residues in its autoinhibitory C terminus. The expression and activity of both P- and V-type H+-ATPases are highly regulated by hormones and environmental cues. In this review, we summarize the recent advances in understanding of the evolution, regulation, and physiological roles of P- and V-type H+-ATPases, which coordinate and are involved in plant growth and stress adaptation. Understanding the different roles and the regulatory mechanisms of P- and V-type H+-ATPases provides a new perspective for improving plant growth and stress tolerance by modulating the activity of H+-ATPases, which will mitigate the increasing environmental stress conditions associated with ongoing global climate change.

Arabidopsis CAMTA3/SR1 is involved in drought stress tolerance and ABA signaling.

Calcium/calmodulin signals are important for various cellular and physiological activities in plants. Calmodulin binding transcription activators also named Signal Responsive (SR) proteins belong to an important calcium/calmodulin-dependent transcription factor family that plays critical roles in stress responses. However, the role of SRs in abscisic acid (ABA) regulated plant responses to drought stress is largely unknown. Here, we characterized the role of Arabidopsis SR1 in drought stress tolerance and ABA response by analyzing the phenotypes of SR1 knockout and SR1-overexpression plants. sr1 mutants which accumulate salicylic acid (SA) were found more sensitive to drought stress and showed a higher water loss rate as compared with wild-type. By contrast, SR1-overexpression lines exhibited increased drought tolerance and less water loss than wild-type. Furthermore, sr1 mutants showed reduced ABA response in seed germination, root elongation, and stomatal closure, while SR1-overexpression lines displayed more sensitive to ABA than wild-type. In addition, the drought-sensitive and ABA-insensitive phenotypes of sr1 mutants were recovered by diminishing SA accumulation via knockouts of SA synthesizer ICS1 or activator PAD4, or through expression of SA-degrading enzyme NahG. Some drought/ABA-responsive genes exhibited differentially expressed in sr1 mutants and SR1-overexpression plants. These results suggest that SR1 plays a positive role in drought stress tolerance and ABA response, and drought/ABA responses are antagonized by SA accumulation that is negatively regulated by SR1.

Molecular regulation of zinc deficiency responses in plants.

Zinc (Zn) is an essential micronutrient for plants and animals. Because of its low availability in arable soils worldwide, Zn deficiency is becoming a serious agricultural problem resulting in decreases of crop yield and nutritional quality. Plants have evolved multiple responses to adapt to low levels of soil Zn supply, involving biochemical and physiological changes to improve Zn acquisition and utilization, and defend against Zn deficiency stress. In this review, we summarize the physiological and biochemical adaptations of plants to Zn deficiency, the roles of transporters and metal-binding compounds in Zn homeostasis regulation, and the recent progresses in understanding the sophisticated regulatory mechanisms of Zn deficiency responses that have been made by molecular and genetic analyses, as well as diverse 'omics' studies. Zn deficiency responses are tightly controlled by multiple layers of regulation, such as transcriptional regulation that is mediated by transcription factors like F-group bZIP proteins, epigenetic regulation at the level of chromatin, and post-transcriptional regulation mediated by small RNAs and alternative splicing. The insights into the regulatory network underlying Zn deficiency responses and the perspective for further understandings of molecular regulation of Zn deficiency responses have been discussed. The understandings of the regulatory mechanisms will be important for improving Zn deficiency tolerance, Zn use efficiency, and Zn biofortification in plants.

Molecular basis of plasma membrane H+-ATPase function and potential application in the agricultural production.

Increase of crop yield is always the desired goal, manipulation of genes in relation to plant growth is a shortcut to promote crop yield. The plasma membrane (PM) H+-ATPase is the plant master enzyme; the energy yielded by ATP hydrolysis pumps H+ out of cells, establishes the membrane potential, maintains pH homeostasis and provides the proton-motive force required for transmembrane transport of many materials. PM H+-ATPase is involved in root nutrient uptake, epidermal stomatal opening, phloem sucrose loading and unloading, and hypocotyl cell elongation. In this review, we summarize the recent progresses in roles of PM H+-ATPase in nutrient uptake and light-induced stomatal opening and discuss the pivotal role of PM H+-ATPase in crop yield improvement and its potential application in agricultural production by modulating the expression of PM H+-ATPase in crops.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants.

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.