An Arabidopsis ABC Transporter Mediates Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Roots

The remodeling of root architecture is a major developmental response of plants to phosphate （Pi） deficiency and is thought to enhance a plant＇s ability to forage for the available Pi in topsoil. The underlying mechanism controlling this response, however, is poorly understood. In this study, we identified an Arabidopsis mutant, hps 10 （hypersensitive to Pi starvation 10）, which is morphologically normal under Pi sufficient condition but shows increased inhibition of primary root growth and enhanced production of lateral roots under Pi defi- ciency, hpslO is a previously identified allele （als3-3） of the ALUMINUM SENSITIVE3 （ALS3） gene, which is involved in plant tolerance to aluminum toxicity. Our results show that ALS3 and its interacting protein AtSTAR1 form an ABC transporter complex in the tonoplast. This protein complex mediates a highly electro- genic transport in Xenopus oocytes. Under Pi deficiency, als3 accumulates higher levels of Fe3＋ in its roots than the wild type does. In Arabidopsis, LPR1 （LOW PHOSPHATE ROOT1） and LPR2 encode ferroxidases, which when mutated, reduce Fe3＋ accumulation in roots and cause root growth to be insensitive to Pi defi- ciency. Here, we provide compelling evidence showing that ALS3 cooperates with LPR1/2 to regulate Pi deficiency-induced remodeling of root architecture by modulating Fe homeostasis in roots.

Blue Light-Triggered Chemical Reactions Underlie Phosphate Deficiency-Induced Inhibition of Root Elongation oM/ab/dops/s Seedlings Grown in Petri Dishes

To tolerate phosphate(Pi)deficiency in the environment,plants alter their developmental and metabolic programs.In the past two decades,researchers have extensively used Petri dish-grown seedlings of the model plant Arabidopsis thaliana to study the molecular mechanisms underlying root developmental responses to Pi deficiency.A typical developmental response of the Petri dish-grown Arabidopsis seedlings to Pi deficiency is the inhibited growth of primary root(PR).This response is generally thought to enhance the production of lateral roots and root hairs,which increases the plant’s ability to obtain Pi and is therefore regarded as an active cellular response.Here,we report that direct illumination of root surface with blue light is critical and sufficient for Pi deficiency-induced inhibition of PR growth in Arabidopsis seedlings.We further show that a blue light-triggered malate-mediated photo-Fenton reaction and a canonical Fenton reaction form an Fe redox cycle in the root apoplast.This Fe redox cycle results in the production of hydroxyl radicals that inhibit PR growth.In addition to revealing the molecular mechanism underlying Pi deficiency-induced inhibition of PR growth,our work demonstrated that this developmental change is not an active cellular response;instead,it is a phenotype resulting from root growth in transparent Petri dishes.This finding is significant because illuminated,transparent Petri dishes have been routinely used to study Arabidopsis root responses to environmental changes.

Ubiquitin-Specific Protease 14 （UBP14） Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis

A mutant isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not caused by a lack of Pi. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Complementation analyses showed that the PER1 gene encodes an ubiquitin-specific protease, UBP14. The mutation caused a synonymous substitution in the 12th exon of this gene, resulting in a lower abundance of the UBP14 protein, probably as a consequence of reduced translation efficiency. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate.

Long-distance blue light signalling regulates phosphate deficiency-induced primary root growth inhibition

Although roots are mainly embedded in the soil, recent studies revealed that light regulates mineral nutrient uptake by roots. However, it remains unclear whether the change in root system architecture in response to different rhizosphere nutrient statuses involves light signaling. Here, we report that blue light regulates primary root growth inhibition under phosphate-deficient conditions through the cryptochromes and their downstream signaling factors. We showed that the inhibition of root elongation by low phosphate requires blue light signal perception at the shoot and transduction to the root. In this process, SPA1 and COP1 play a negative role while HY5 plays a positive role. Further experiments revealed that HY5 is able to migrate from the shoot to root and that the shoot-derived HY5 autoactivates root HY5 and regulates primary root growth by directly activating the expression of LPR1, a suppressor of root growth under phosphate starvation. Taken together, our study reveals a regulatory mechanism by which blue light signaling regulates phosphate deficiency-induced primary root growth inhibition, providing new insights into the crosstalk between light and nutrient signaling.

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.

Identification of two glycerophosphodiester phosphodiesterase genes in maize leaf phosphorus remobilization

Phosphate deficiency is one of the leading causes of crop productivity loss.Phospholipid degradation liberates phosphate to cope with phosphate deficiency.Glycerophosphodiester phosphodiesterases(GPX-PDEs)hydrolyse the intermediate products of phospholipid catabolism glycerophosphodiesters into glycerol-3-phosphate,a precursor of phosphate.However,the function of GPX-PDEs in phosphate remobilization in maize remains unclear.In the present study,we characterized two phosphate deficiency-inducible GPX-PDE genes,ZmGPX-PDE1 and ZmGPX-PDE5,in maize leaves.ZmGPX-PDE1 and ZmGPX-PDE5 were transcriptionally regulated by ZmPHR1,a well-described phosphate starvation-responsive transcription factor of the MYB family.Complementation of the yeast GPX-PDE mutant gde1Δindicated that ZmGPX-PDE1 and ZmGPX-PDE5 functioned as GPX-PDEs,suggesting their roles in phosphate recycling from glycerophosphodiesters.In vitro enzyme assays showed that ZmGPX-PDE1 and ZmGPX-PDE5 catalysed glycerophosphodiester degradation with different substrate preferences for glycerophosphoinositol and glycerophosphocholine,respectively.ZmGPX-PDE1 was upregulated during leaf senescence,and more remarkably,loss of ZmGPXPDE1 inmaize compromised the remobilization of phosphorus fromsenescing leaves to young leaves,resulting in a stay-green phenotype under phosphate starvation.These results suggest that ZmGPX-PDE1 catalyses the degradation of glycerophosphodiesters in maize,promoting phosphate recycling from senescing leaves to new leaves.This mechanism is crucial for improving phosphorus utilization efficiency in crops.

Brassinosteroid signaling regulates phosphate starvation-induced malate secretion in plants

Inorganic phosphate(Pi)is often limited in soils due to precipitation with iron(Fe)and aluminum(Al).To scavenge heterogeneously distributed phosphorus(P)resources,plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe-P or Al-P oxides.In this study,we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE-RESISTANT 1(BZR1)expression in Arabidopsis thaliana.Exogenous 2,4-epibrassinolide treatment or constitutive activation of BZR1(in the bzr1-D mutant)significantly reduced primary root growth inhibition under Pi-starvation conditions by downregulating ALUMINUM-ACTIVATED MALATE TRANSPORTER 1(ALMT1)expression and malate secretion.Furthermore,At BZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1(STOP1)on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis.The ratio of nuclear-localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis.In addition,BZR1-inhibited malate secretion is conserved in rice(Oryza sativa).Our findings provide insight into plant mechanisms for optimizing the secretion of malate,an important carbon resource,to adapt to Pi-deficiency stress.

Overexpressing HRS1 Confers Hypersensitivity to Low Phosphate-Elicited Inhibition of Primary Root Growth in Arabidopsis thaliana

Phosphate （Pi） deficiency causes dramatic root system architecture （RSA） changes in higher plants. Here we report that overexpression of HRS1 leads to enhanced sensitivity to low Pi-elicited inhibition of primary root growth in Arabidopsis thaliana seedlings. Bioinformatic investigations uncovered that HRS1 and its six homologs encode putative G2-like transcription factors in Arabidopsis. Analysis of promoter：：GUS reporter lines revealed that HRS1 transcripts were present mainly in the root hair region and root hair cells under Pi-sufficient conditions. Pi deprivation increased HRS1 expression level and expanded its expression domain. Although HRS1 knockout mutant did not differ from wild type （WT） control irrespective of Pi status, its overexpression lines were significantly more susceptible to low Pi-elicited primary root shortening. In both WT and HRS1 overexpression seedlings, low Pi-induced primary root shortening was accompanied by enhanced root hair cell differentiation, but this enhancement occurred to a greater extent in the latter genotype. Collectively, our data suggest that HRS1 may be involved in the modulation of primary root and root hair growth in Pi-deprived Arabidopsis seedlings, and provide useful clues for further research into the function of HRS1 and its homologs and the mechanisms behind RSA changes under Pi-deficient conditions.

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield

Phosphorus is a major nutrient vital for plant growth and development,with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids.Here,we report that NON-SPECIFIC PHOSPHOLIPASE C4(NPC4)in rapeseed(Brassica napus)releases phosphate from phospholipids to promote growth and seed yield,as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions.Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated nuclease 9(Cas9)-mediated knockout of Bna NPC4 led to elevated accumulation of phospholipids and decreased growth,whereas overexpression(OE)of Bna NPC4resulted in lower phospholipid contents and increased plant growth and seed production.We demonstrate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro,and plants with altered Bna NPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots,with a greater change in glycerolipids than sphingolipids in leaves,particularly under phosphate deficiency conditions.In addition,Bna NPC4-OE plants led to the upregulation of genes involved in lipid metabolism,phosphate release,and phosphate transport and an increase in free inorganic phosphate in leaves.These results indicate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.

Plant Phosphatidylcholine-Hydrolyzing Phospholipases C NPC3 and NPC4 with Roles in Root Development and Brassinolide Signaling in Arabidopsis thaliana

Phosphatidylcholine-hydrolyzing phospholipase C （PC-PLC） catalyzes the hydrolysis of phosphatidylcholine （PC） to generate phosphocholine and diacylglycerol （DAG）. PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C （PI-PLC）. Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes （NPC1 to NPC6） were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated PNPc3：GUS and PNPc4：GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated （BL） seedlings. PNPc4：GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 I^M BL BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 I^M BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man

Objective To detect new mutations among 29 glucose 6 phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province Methods The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing Results Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified Nine cases remained to be defined The G1381A mutation is a novel mis sense mutation, with a substitution of threonine for alanine (A461T) The resultant G6PD had reduced enzymatic activity In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation Conclusion A novel mis sense mutation G1381A was found This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity The loss of the Stu I restriction site offers a rapid method for the detection of this mutation

Source of inorganic N affects the cost of growth in a legume tree species(Virgilia divaricata)from the Mediterrean-type Fynbos ecosystem

Aims In Mediterranean-type ecosystem,the Cape Fynbos,legumes may be able to switch between soil N and atmospheric N_(2) sources during growth to adjust the carbon costs of N acquisition.This study investigated the utilization of different inorganic N sources by Virgilia divaricata,a native legume from the Mediterranean-type ecosystem of the Cape Floristic Region.Methods Plants were cultivated in sterile quartz sand,supplied with 25%strength Long Ashton nutrient solution,modified to contain 500μM Phosphate.At the phosphate level(500μM),plants were treated with 500μM NH_(4)NO_(3)(treatment named N),or grown in N-free nutrient solution and inoculated with effective Burkholderia sp.(Bact.)or treated with combined N sources(500μM NH_(4)NO_(3))and inoculated with effective Burkholderia sp.(N+Bact.).Important Findings The application of NH_(4)NO_(3) to the legumes resulted in a greater increase in plant dry matter.Carbon construction costs were higher in plants that were supplied with mineral and symbiotic N sources.Maximum photosynthetic rates per leaf area was maintained,irrespective of the N sources.Although the plant roots were nodulated,the plant dependence on N_(2) fixation decreased with addition of N.Roots and nodules of the plants solely reliant on N_(2) fixation showed an increase in glutamine content.These results show that V.divaricata is highly adapted for growth at the forest margin.Fynbos and possibly anthropic soils by utilizing both atmospheric and soil N sources.