草地早熟禾Phosphate Starvation Response 2基因的克隆及表达分析

磷饥饿响应(Phosphate starvation response PHR2)家族基因在植物磷(Pi)信号调节网络中发挥重要指示作用。为了解该基因在逆境胁迫中的反应机制,本研究从草地早熟禾(Poa pratensis L.)中克隆了PHR2基因,对其进行生物信息学和基因表达模式分析,并分析PHR2基因在草地早熟禾逆境胁迫中的作用。结果表明:草地早熟禾PHR2属于MYB-CC型转录因子,与节节麦氨基酸序列高度同源,该基因主要定位在细胞核。PHR2基因在草地早熟禾的根、茎、叶、穗中均有表达,其中根和穗中表达高于茎和叶;低磷诱导叶中PHR2基因表达低于适磷下该基因表达;干旱胁迫抑制该基因在根、叶的表达,但根与叶的转录调控模式存在差异。本研究为探究PHR家族基因在逆境胁迫中的功能提供一定的理论依据。

GmPHR1, a Novel Homolog of the AtPHR1 Transcription Factor, Plays a Role in Plant Tolerance to Phosphate Starvation

GmPHR1 from soybean （Glycine max） was isolated and characterized. This novel homolog of the AtPHR1 transcription factor confers tolerance to inorganic phosphate （Pi）-starvation. The gene is 2 751 bp long, with an 819-bp open reading frame and ifve introns. Analysis of transcription activity in yeast revealed that the full-length GmPHR1 and its C-terminal activate the reporter genes for His, Ade and Ura, suggesting that the C-terminal peptide functions as a transcriptional activator. Quantitative real-time PCR indicated that patterns of GmPHR1 expression differed. For example, under low-Pi stress, this gene was quickly induced in the tolerant JD11 after 0.5 h, with expression then decreasing slowly before peaking at 12-24 h. By contrast, induction in the sensitive Niumaohuang （NMH） was slow, peaking at 6 h before decreasing quickly at 9 h. GmPHR1 showed sub-cellular localization in the nuclei of onion epidermal cells and Arabidopsis roots. Growth parameters in wild-type （WT） Arabidopsis plants as well as in overexpression （OE） transgenic lines were examined. Under low-Pi conditions, values for shoot, root and whole-plant dry weights, root to shoot ratios, and lengths of primary roots were signiifcantly greater in OE lines than in the WT. These data demonstrate that GmPHR1 has an important role in conferring tolerance to phosphate starvation.

TaMADS2-3D,a MADS transcription factor gene,regulates phosphate starvation responses in plants

Soil inorganic phosphate(Pi)levels are frequently suboptimal for the growth and development of crop plants.Although MADS-box genes participate in diverse plant developmental processes,their involvement in phosphate starvation responses(PSRs)remains unclear.We identified a type I MADS-box transcription factor gene,Ta MADS2-3 D,which was rapidly induced under low-Pi stress in roots of wheat(Triticum aestivum).A Ta MADS2-3 D-GFP fusion protein was found located in the nucleus.Transgenic Arabidopsis plants overexpressing Ta MADS2-3 D(Ta MADS2-3 DOE)showed shortened primary roots,increased lateral root density,and retarded seedling growth under high-Pi(HP)conditions,accompanied by increased Pi contents in their shoots and roots.The Arabidopsis Ta MADS2-3 DOE plants showed similar PSR phenotypes under low Pi(LP)conditions.These results indicate constitutive activation of PSRs by overexpression of Ta MADS2-3 D in Arabidopsis.Reactive oxygen species(ROS),H_(2)O_(2)and O_(2)^(-),levels were increased in root tips of Arabidopsis Ta MADS2-3 DOE plants under HP conditions.Transcriptome analysis of Arabidopsis Ta MADS2-3 DOE plants under different Pi regimes revealed expression changes for a variety of PSR genes including At ZAT6.Overexpression of Ta MADS2-3 D in wheat also led to constitutive activation of PSRs.We propose that Ta MADS2-3 D regulates plant PSRs probably by modulating ROS homeostasis,root development,PSR gene expression,and Pi uptake.This study increases our understanding of plant PSR regulation and provides a valuable gene for improving phosphorus-use efficiency in wheat and other crops.

EoPHR2,a Phosphate Starvation Response Transcription Factor,Is Involved in Improving Low-Phosphorus Stress Resistance in Eremochloa ophiuroides

As a macronutrient,Phosphorus(P)takes many roles in plant growth and development.It should be significant to explore the molecular mechanism of low-phosphorus stress response of plants.Phosphate starvation response(PHR)transcription factors play important roles in response to phosphorus deficiency stress in plants.In this study,we isolated a gene related to the plant phosphorus signaling system from the acid-soil-resistant centipedegrass(Eremochloa ophiuroides[Munro]Hack.),termed EoPHR2.The subcellular localization of EoPHR2 protein was observed to be nuclear located.The expression patterns of EoPHR2 in different tissues and Al/Pi-stress conditions were analyzed by qRT-PCR,they suggested a potential role in response to the multiple-stress under acid soil adversity.Based on the functional identification through transgenic plants,we found that(1)EoPHR2 is involved in the Pi-signaling pathway,and(2)overexpression of EoPHR2 mimics Pi-starvation signalling resulting on enhanced roots whether under Pi-deficiency stress or not.In conclusion,EoPHR2 transcription factor plays a role in response to the multiple stresses under acid soil conditions,improving the low-phosphorus stress resistance of Eremochloa ophiuroides.

Signaling Components Involved in Plant Responses to Phosphate Starvation

Phosphorus is one of the macronutrients essential for plant growth and development. Many soils around the world are deficient in phosphate （Pi） which is the form of phosphorus that plants can absorb and utilize. To cope with the stress of Pi starvation, plants have evolved many elaborate strategies to enhance the acquisition and utilization of Pi from the environment. These strategies include morphological, biochemical and physiological responses which ultimately enable plants to better survive under low Pi conditions. Though these adaptive responses have been well described because of their ecological and agricultural importance, our studies on the molecular mechanisms underlying these responses are still in their infancy. In the last decade, significant progresses have been made towards the identification of the molecular components which are involved in the control of plant responses to Pi starvation. In this article, we first provide an overview of some major responses of plants to Pi starvation, then summarize what we have known so far about the signaling components involved in these responses, as well as the roles of sugar and phytohormones.

Primary root and root hair development regulation by OsAUX4 and its participation in the phosphate starvation response

Among the five members of AUX1/LAX genes coding for auxin carriers in rice,only OsAUX1 and OsAUX3 have been reported.To understand the function of the other AUX1/LAX genes,two independent alleles of osaux4 mutants,osaux4-1 and osaux4-2,were constructed using the CRISPR/Cas9 editing system.Homozygous osaux4-1 or osaux4-2 exhibited shorter primary root(PR)and longer root hair(RH)compared to the wild-type Dongjin(WT/DJ),and lost response to indoleacetic acid(IAA)treatment.OsAUX4 is intensively expressed in roots and localized on the plasma membrane,suggesting that OsAUX4 might function in the regulation of root development.The decreased meristem cell division activity and the downregulated expression of cell cycle genes in root apices of osaux4 mutants supported the hypothesis that OsAUX4 positively regulates PR elongation.OsAUX4 is expressed in RH,and osaux4 mutants showing longer RH compared to WT/DJ implies that OsAUX4 negatively regulates RH development.Furthermore,osaux4 mutants are insensitive to Pi starvation(-Pi)and OsAUX4 effects on the-Pi response is associated with altered expression levels of Pi starvation-regulated genes,and auxin distribution/contents.This study revealed that OsAUX4 not only regulates PR and RH development but also plays a regulatory role in crosstalk between auxin and-Pi signaling.

Roles of Ubiquitination in the Control of Phosphate Starvation Responses in Plants

Throughout evolution, plants have evolved sophisticated adaptive responses that allow them to grow with a limited supply of phos-phate, the preferential form in which the essential macronutrient phosphorus is absorbed by plants. Most of these responses are aimed to increase phosphate availability and acquisition through the roots, to optimize its usage in metabolic processes, and to protect plants from the deleterious effects of phosphate deficiency stress. Regulation of these adaptive responses requires fine percep- tion of the external and internal phosphate levels, and a complex signal transduction pathway that integrates information on the phosphate status at the whole-plant scale. The molecular mecha-nisms that participate in phosphate homeostasis include transcriptional control of gene expression, RNA silencing mediated by microRNAs, regulatory non-coding RNAs of miRNA activity, phosphate transporter trafficking, and post-translational modification of proteins, such as phosphorylation, sumoylation and ubiquitination. Such a varied regulatory repertoire reflects the complexity intrinsic to phosphate surveying and signaling pathways. Here, we describe these regulatory mechanisms, emphasizing the increasing importance of ubiquitination in the control of phosphate starvation responses.

Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in Bacillus licheniformis

Extracellular polymeric substances(EPSs)are extracellular macromolecules in bacteria,which function in cell growth and show potential for mechanism study and biosynthesis application.However,the biosynthesis mechanism of EPS is still not clear.We herein chose Bacillus licheniformis CGMCC 2876 as a target strain to investigate the EPS biosynthesis.epsK,a member of eps cluster,the predicted polysaccharide synthesis cluster,was overexpressed and showed that the overexpression of epsK led to a 26.54%decrease in the production of EPS and resulted in slenderer cell shape.Transcriptome analysis combined with protein-protein interactions analysis and protein modeling revealed that epsK was likely responsible for the synthesis of teichuronic acid,a substitute cell wall component of teichoic acid when the strain was suffering phosphate limitation.Further cell cultivation showed that either phosphate limitation or the overexpression of teichuronic acid synthesis genes,tuaB and tuaE could similarly lead to EPS reduction.The enhanced production of teichuronic acid induced by epsK overexpression triggered the endogenous phosphate starvation,resulting in the decreased EPS synthesis and biomass,and the enhanced bacterial chemotaxis.This study presents an insight into the mechanism of EPS synthesis and offers the potential in controllable synthesis of target products.

The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis

Plasticity in root system architecture(RSA)allows plants to adapt to changing nutritional status in the soil.Phosphorus availability is a major determinant of crop yield,and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition.Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants,whether and how epigenetic regulatory mechanisms contribute is poorly understood.Here,we report that the Switch defective/sucrose non-fermentable(SWI/SNF)ATPase BRAHMA(BRM)is involved in the local response to phosphate(Pi)starvation.The loss of BRM function induces iron(Fe)accumulation through increased LOW PHOSPHATE ROOT1(LPR1)and LPR2 expression,reducing primary root length under Pi deficiency.We also demonstrate that BRM recruits the histone deacetylase(HDA)complex HDA6-HDC1 to facilitate histone H3 deacetylation at LPR loci,thereby negatively regulating local Pi deficiency responses.BRM is degraded under Pi deficiency conditions through the 26 S proteasome pathway,leading to increased histone H3 acetylation at the LPR loci.Collectively,our data suggest that the chromatin remodeler BRM,in concert with HDA6,negatively regulates Fe-dependent local Pi starvation responses by transcriptionally repressing the RSA-related genes LPR1 and LPR2 in Arabidopsis thaliana.

Influences of external nutrient conditions on the transcript levels of a nitrate transporter gene in Skeletonema costatum

To verify the feasibility of high-affinity nitrate transporter gene （Nrt2） as an indicator of nitrogen status, changes in the transcript levels of transcripts associated with phosphate starvation and different nitrate concentrations were studied using real-time quantitative reverse-transcription PCR （QRT-PCR） technology in batch cultures of Skeletonema costatum. The results show that compared with P-replete condition, P starvation could reduce the Nrt2 transcript levels apparently. Nrt2 transcript levels had a significant negative linear correlation with nitrate concentrations below 40 pmol/L. The results of 48 h short-term incubation experiment under different nitrate concentrations confirmed this correlation, and the following regression equation is built： y = -3.305x ＋ 98.95, R2 = 0.988, where x represents nitrate concentrations （〈40 btmol/L） and y represents the Nrt2 transcript levels.

Complex Regulation of Plant Phosphate Transporters and the Gap between Molecular Mechanisms and Practical Application： What Is Missing？

It has been almost 25 years since the first report of the gene encoding a high-affinity phosphate transporter （PT）, PH084, in yeast. Since then, an increasing number of yeast PH084 homologs as well as other genes encoding proteins with phosphate （Pi） transport activities have been identified and functionally characterized in diverse plant species. Great progress has been made also in deciphering the molecular mechanism underlying the regulation of the abundance and/or activity of these genes and their products. The regulatory genes affect plant Pi homeostasis commonly through direct or indirect regulation of the abundance of PTs at different levels. However, little has been achieved in the use of PTs for developing genetically modified crops with high phosphorus use efficiency （PUE）. This might be a consequence of overemphasizing Pi uptake from the rhizosphere and lack of knowledge about the roles of PTs in Pi transport and recycling within the plant that are required to optimize PUE. Here, we mainly focused on the genes encoding proteins with Pi transport activities and the emerging understanding of their regulation at the transcriptional, posttranscriptional, translational, and post-translational levels. In addition, we propose potential strategies for effective use of PTs in improving plant growth and development.

Comparative genetic analysis of Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

Induction and secretion of acid phosphatases （APases） is thought to be an adaptive mechanism that helps plants survive and grow under phosphate （Pi） deprivation, in Arabidopsis, there are 29 purple acid phosphatase （AtPAP） genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAPlo is mainly a secreted APase. On Pi-deficient （P-） medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate （Fru-6-P）, growth of atpaplo was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type （WT）. Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.

Identification of micro RNAs in six solanaceous plants and their potential link with phosphate and mycorrhizal signalings

To date, only a limited number of solanaceous miRNAs have been deposited in the miRNA database. Here,Rgenome-wide bioinformatic identification of miRNAs was performed in six solanaceous plants（potato, tomato, tobacco,eggplant, pepper, and petunia）. A total of 2,239 miRNAs were identified following a range of criteria, of which 982 were from potato, 496 from tomato, 655 from tobacco, 46 from eggplant,45 were from pepper, and 15 from petunia. The sizes of miRNA families and miRNA precursor length differ in all the species.Accordingly, 620 targets were predicted, which could be functionally classified as transcription factors, metabolic enzymes, RNA and protein processing proteins, and other proteins for plant growth and development. We also showed evidence for miRNA clusters and sense and antisense miR NAs.Additionally, five Pi starvation- and one arbuscular mycorrhiza（AM）-related cis-elements were found widely distributed in the putative promoter regions of the miRNA genes. Selected miRNAs were classified into three groups based on the presence or absence of P1BS and MYCScis-elements, and their expression in response to Pi starvation and AM symbiosis was validated by quantitative reverse transcription-polymerase chain reaction（qRT-PCR）. These results show that conserved miRNAs exist in solanaceous species and they might play pivotal roles in plant growth, development, and stress responses.

The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis

PHR1(PHOSPHATE STARVATION RESPONSE1)plays key roles in the inorganic phosphate(Pi)starvation response and in Pi deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear,and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi deficiency-induced anthocyanin accumulation in apple(Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1,leading to anthocyanin accumulation. In addition,the E3 ubiquitin ligase SEVEN IN ABSENTIA1(MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1.Moreover, the protein kinase apple BRASSINOSTEROID INSENSITIVE2(MdBIN2) phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together,these findings not only demonstrate the regulatory role of MdPHR1 in Pi starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1.

Arsenite provides a selective signal that coordinates arsenate uptake and detoxification through the regulation of PHR1 stability in Arabidopsis

In nature,plants acquire nutrients from soils to sustain growth,and at the same time,they need to avoid the uptake of toxic compounds and/or possess tolerance systems to cope with them.This is particularly challenging when the toxic compound and the nutrient are chemically similar,as in the case of phosphate and arsenate.In this study,we demonstrated that regulatory elements of the phosphate starvation response(PSR)coordinate the arsenate detoxification machinery in the cell.We showed that arsenate repression of the phosphate transporter PHT1;1 is associated with the degradation of the PSR master regulator PHR1.Once arsenic is sequestered into the vacuole,PHR1 stability is restored and PHT1;1 expression is recovered.Furthermore,we identified an arsenite responsive SKP1-like protein and a PHR1 interactor F-box(PHIF1)as constituents of the SCF complex responsible for PHR1 degradation.We found that arsenite,the form to which arsenate is reduced for compartmentalization in vacuoles,represses PHT1;1 expression,providing a highly selective signal versus phosphate to control PHT1;1 expression in response to arsenate.Collectively,our results provide molecular insights into a sensing mechanism that regulates arsenate/phosphate uptake depending on the plant’s detoxification capacity.