The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high‐phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO₂ fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non‐photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton‐motive force across thylakoids. Small‐angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long‐range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild‐type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro‐organization of complexes and induction of photoprotective mechanisms.     

GTPase ROP6 negatively modulates phosphate deficiency through inhibition of PHT1;1 and PHT1;4 in Arabidopsis thaliana

Phosphorus, an essential macroelement for plant growth and development, is a major limiting factor for sustainable crop yield. The Rho of plant (ROP) GTPase is involved in regulating multiple signal transduction processes in plants, but potentially including the phosphate deficiency signaling pathway remains unknown. Here, we identified that the rop6 mutant exhibited a dramatic tolerant phenotype under Pi-deficient conditions, with higher phosphate accumulation and lower anthocyanin content. In contrast, the rop6 mutant was more sensitive to arsenate (As(V)) toxicity, the analog of Pi. Immunoblot analysis displayed that the ROP6 protein was rapidly degraded through ubiquitin/26S proteasome pathway under Pi-deficient conditions. In addition, pull-down assay using GST-RIC1 demonstrated that the ROP6 activity was decreased obviously under Pi-deficient conditions. Strikingly, protein–protein interaction and two-voltage clamping assays demonstrated that ROP6 physically interacted with and inhibited the key phosphate uptake transporters PHT1;1 and PHT1;4 in vitro and in vivo. Moreover, genetic analysis showed that ROP6 functioned upstream of PHT1;1 and PHT1;4. Thus, we conclude that GTPase ROP6 modulates the uptake of phosphate by inhibiting the activities of PHT1;1 and PHT1;4 in Arabidopsis.     

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.     

A chloroplast phosphate transporter,PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses

The uptake and distribution of Pi in plants requires multiple Pi transport systems that must function in concert to maintain homeostasis throughout growth and development. The Pi transporter PHT2;1 of Arabidopsis shares similarity with members of the Pi transporter family, which includes Na(+)/Pi symporters of fungal and animal origin and H(+)/Pi symporters of bacterial origin. Sequence comparisons between proteins of this family revealed that plant members possess extended N termini, which share features with chloroplast transit peptides. Localization of a PHT2;1-green fluorescent protein fusion protein indicates that it is present in the chloroplast envelope. A Pi transport function for PHT2;1 was confirmed in yeast using a truncated version of the protein lacking its transit peptide, which allowed targeting to the plasma membrane. To assess the in vivo role of PHT2;1 in phosphorus metabolism, we identified a null mutant, pht2;1-1. Analysis of the mutant reveals that PHT2;1 activity affects Pi allocation within the plant and modulates Pi-starvation responses, including the expression of Pi-starvation response genes and the translocation of Pi within leaves.     

ABI4 downregulates expression of the sodium transporter HKT1;1 in Arabidopsis roots and affects salt tolerance

A plant's ability to cope with salt stress is highly correlated with their ability to reduce the accumulation of sodium ions in the shoot. Arabidopsis mutants affected in the ABSCISIC ACID INSENSITIVE (ABI) 4 gene display increased salt tolerance, whereas ABI4‐overexpressors are hypersensitive to salinity from seed germination to late vegetative developmental stages. In this study we demonstrate that abi4 mutant plants accumulate lower levels of sodium ions and higher levels of proline than wild‐type plants following salt stress. We show higher HKT1;1 expression in abi4 mutant plants and lower levels of expression in ABI4‐overexpressing plants, resulting in reduced accumulation of sodium ions in the shoot of abi4 mutants. HKT1;1 encodes a sodium transporter which is known to unload sodium ions from the root xylem stream into the xylem parenchyma stele cells. We have shown recently that ABI4 is expressed in the root stele at various developmental stages and that it plays a key role in determining root architecture. Thus ABI4 and HKT1;1 are expressed in the same cells, which suggests the possibility of direct binding of ABI4 to the HKT1;1 promoter. In planta chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that ABI4 binds two highly related sites within the HKT1;1 promoter. These sites, GC(C/G)GCTT(T), termed ABI4‐binding element (ABE), have also been identified in other ABI4‐repressed genes. We therefore suggest that ABI4 is a major modulator of root development and function.     

Lack of the Golgi phosphate transporter PHT4;6 causes strong developmental defects,constitutively activated disease resistance mechanisms and altered intracellular phosphate compartmentation in Arabidopsis

The Golgi‐located phosphate exporter PHT4;6 has been described as involved in salt tolerance but further analysis on the physiological impact of PHT4;6 remained elusive. Here we show that PHT4;6–GFP is targeted to the trans‐Golgi compartment and that loss of function of this carrier protein has a dramatic impact on plant growth and development. Knockout mutants of pht4;6 exhibit a dwarf phenotype that is complemented by the homologous gene from rice (Oryza sativa). Interestingly, pht4;6 mutants show altered characteristics of several Golgi‐related functions, such as an altered abundance of certain N‐glycosylated proteins, altered composition of cell‐wall hemicelluose, and higher sensitivity to the Golgi α‐mannosidase and the retrograde transport inhibitors kifunensine and brefeldin A, respectively. Moreover, pht4;6 mutants exhibit a ‘mimic disease’ phenotype accompanied by constitutively activated pathogen defense mechanisms and increased resistance against the virulent Pseudomonas syringae strain DC3000. Surprisingly, pht4;6 mutants also exhibit phosphate starvation symptoms, as revealed at the morphological and molecular level, although total Pi levels in wild‐type and pht4;6 plants are similar. This suggested that subcellular Pi compartmentation was impaired. By use of nuclear magnetic resonance (NMR), increased Pi concentration was detected in acidic compartments of pht4;6 mutants. We propose that impaired Pi efflux from the trans‐Golgi lumen results in accumulation of inorganic phosphate in other internal compartments, leading to low cytoplasmic phosphate levels with detrimental effects on plant performance.     

A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice

A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, (33)P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions.     

Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies. Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.     

Integrated dominance mechanisms regulate reproductive architecture in Arabidopsis thaliana and Brassica napus

The production of seed in flowering plants is complicated by the need to first invest in reproductive shoots, inflorescences, flowers, and fruit. Furthermore, in many species, it will be months between plants generating flowers and setting seed. How can plants therefore produce an optimal seed-set relative to environmental resources when the “reproductive architecture” that supports seed-set needs to be elaborated so far in advance? Here, we address this question by investigating the spatio-temporal control of reproductive architecture in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We show that resource and resource-related signals such as substrate volume play a key role in determining the scale of reproductive effort, and that this is reflected in the earliest events in reproductive development, which broadly predict the subsequent reproductive effort. We show that a series of negative feedbacks both within and between developmental stages prevent plants from over-committing to early stages of development. These feedbacks create a highly plastic, homeostatic system in which additional organs can be produced in the case of reproductive failure elsewhere in the system. We propose that these feedbacks represent an “integrated dominance” mechanism that allows resource use to be correctly sequenced between developmental stages to optimize seed set.     

An APETALA1 ortholog affects plant architecture and seed yield component in oilseed rape (Brassica napus L.)

Exploring the natural occurring genetic variation of the wild barley genepool has become a major target of barley crop breeding programmes aiming to increase crop productivity and sustainability in global climate change scenarios. However this diversity remains unexploited and effective approaches are required to investigate the benefits that unadapted genomes could bring to crop improved resilience. In the present study, a set of Recombinant Chromosome Substitution Lines (RCSLs) derived from an elite barley cultivar ‘Harrington’ as the recurrent parent, and a wild barley accession from the Fertile Crescent ‘Caesarea 26–24’, as the donor parent (Matus et al. Genome 46:1010–23, 2003) have been utilised in field and controlled conditions to examine the contribution of wild barley genome as a source of novel allelic variation for the cultivated barley genepool. Twenty-eight RCSLs which were selected to represent the entire genome of the wild barley accession, were genotyped using the 9 K iSelect SNP markers (Comadran et al. Nat Genet 44:1388–92, 2012) and phenotyped for a range of morphological, developmental and agronomic traits in 2 years using a rain-out shelter with four replicates and three water treatments. Data were analysed for marker traits associations using a mixed model approach. We identified lines that differ significantly from the elite parent for both qualitative and quantitative traits across growing seasons and water regimes. The detailed genotypic characterisation of the lines for over 1800 polymorphic SNP markers and the design of a mixed model analysis identified chromosomal regions associated with yield related traits where the wild barley allele had a positive response increasing grain weight and size. In addition, variation for qualitative characters, such as the presence of cuticle waxes on the developing spikes, was associated with the wild barley introgressions. Despite the coarse location of the QTLs, interesting candidate genes for the major marker-trait associations were identified using the recently released barley genome assembly. This study has highlighted the role of exotic germplasm to contribute novel allelic variation by using an optimised experimental approach focused on an exotic genetic library. The results obtained constitute a step forward to the development of more tolerant and resilient varieties.     

Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants

Background and aims

Whereas the expression patterns and kinetic properties of the rice (Oryza sativa) phosphate transporter gene OsPht1; 6 (OsPT6) are well documented, little is known about the biological functions of this gene. The aim of this study was to investigate the roles of OsPT6 in inorganic phosphate (Pi) acquisition and mobilization, and examine its potential to enhance agricultural production.

Methods

Here, we generated OsPT6 overexpression transgenic plants using Wuyujing 7, a widely cultivated variety of japonica rice, and then treated transgenic lines and wild type with different Pi supply in hydroponic and soil experiments to explore the functions of OsPT6 in rice.

Results

The OsPT6-overexpressing rice lines grew better and accumulated more biomass than wild-type plants, and exhibited significant increases in P accumulation in various tissues, including reproductive tissues under both hydroponic and soil culture conditions. Phosphate-uptake experiment using radiolabeled Pi (³³P) showed that the rate of Pi uptake was 75 % and 73 % greater in transgenic plants grown under Pi-sufficient and -deficient conditions, respectively, than the wild-type controls, and that the shoot/root ratio of ³³P was 104 % and 42 % greater, respectively. In addition, the grain yield per transgenic plant was much higher than that of the wild-type plants under field conditions.

Conclusions

Taken together, our results demonstrate that OsPT6 plays a vital role in Pi acquisition and mobilization in rice and suggest that this gene may be used for genetic engineering rice plants that require less Pi fertilizer.     

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10–40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.