The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content.

Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.

Arabidopsis MYB-Related HHO2 Exerts a Regulatory Influence on a Subset of Root Traits and Genes Governing Phosphate Homeostasis.

Phosphate (Pi), an essential macronutrient required for growth and development of plants, is often limiting in soils. Pi deficiency modulates the expression of Pi starvation-responsive (PSR) genes including transcription factors (TFs). Here, we elucidated the role of the MYB-related TF HYPERSENSITIVITY TO LOW PHOSPHATE-ELICITED PRIMARY ROOT SHORTENING1 HOMOLOG2 (HHO2, At1g68670) in regulating Pi acquisition and signaling in Arabidopsis thaliana HHO2 was specifically and significantly induced in different tissues in response to Pi deprivation. Transgenic seedlings expressing 35S::GFP::HHO2 confirmed the localization of HHO2 to the nucleus. Knockout mutants of HHO2 showed significant reduction in number and length of first- and higher-order lateral roots and Pi content of different tissues compared with the wild-type irrespective of the Pi regime. In contrast, HHO2-overexpressing lines exhibited augmented lateral root development, enhanced Pi uptake rate and higher Pi content in leaf compared with the wild-type. Expression levels of PSR genes involved in Pi sensing and signaling in mutants and overexpressors were differentially regulated as compared with the wild-type. Attenuation in the expression of HHO2 in the phr1 mutant suggested a likely influence of PHR1 in HHO2-mediated regulation of a subset of traits governing Pi homeostasis.

Ethylene Response Factor070 regulates root development and phosphate starvation-mediated responses.

Inorganic phosphate (Pi) availability is a major factor determining growth and consequently the productivity of crops. However, it is one of the least available macronutrients due to its high fixation in the rhizospheres. To overcome this constraint, plants have developed adaptive responses to better acquire, utilize, and recycle Pi. Molecular determinants of these adaptive mechanisms include transcription factors (TFs) that play a major role in transcriptional control, thereby regulating genome-scale networks. In this study, we have characterized the biological role of Arabidopsis thaliana Ethylene Response Factor070 (AtERF070), a Pi starvation-induced TF belonging to the Apetala2/Ethylene Response Factor family of TFs in Arabidopsis (Arabidopsis thaliana). It is localized to the nucleus and induced specifically in Pi-deprived roots and shoots. RNA interference-mediated suppression of AtERF070 led to augmented lateral root development resulting in higher Pi accumulation, whereas there were reductions in both primary root length and lateral root number in 12-d-old transgenic seedlings overexpressing AtERF070. When the overexpressing lines were grown to maturity under greenhouse conditions, they revealed a stunted bushy appearance that could be rescued by gibberellic acid application. Furthermore, a number of Pi starvation-responsive genes were modulated in AtERF070-overexpressing and RNA interference lines, thereby suggesting a potential role for this TF in maintaining Pi homeostasis.

Transcriptional regulation of phosphate acquisition by higher plants.

Phosphorus (P), an essential macronutrient required for plant growth and development, is often limiting in natural and agro-climatic environments. To cope with heterogeneous or low phosphate (Pi) availability, plants have evolved an array of adaptive responses facilitating optimal acquisition and distribution of Pi. The root system plays a pivotal role in Pi-deficiency-mediated adaptive responses that are regulated by a complex interplay of systemic and local Pi sensing. Cross-talk with sugar, phytohormones, and other nutrient signaling pathways further highlight the intricacies involved in maintaining Pi homeostasis. Transcriptional regulation of Pi-starvation responses is particularly intriguing and involves a host of transcription factors (TFs). Although PHR1 of Arabidopsis is an extensively studied MYB TF regulating subset of Pi-starvation responses, it is not induced during Pi deprivation. Genome-wide analyses of Arabidopsis have shown that low Pi stress triggers spatiotemporal expression of several genes encoding different TFs. Functional characterization of some of these TFs reveals their diverse roles in regulating root system architecture, and acquisition and utilization of Pi. Some of the TFs are also involved in phytohormone-mediated root responses to Pi starvation. The biological roles of these TFs in transcriptional regulation of Pi homeostasis in model plants Arabidopsis thaliana and Oryza sativa are presented in this review.

Characterization of the phosphate starvation-induced glycerol-3-phosphate permease gene family in Arabidopsis.

Phosphate (Pi) deficiency is one of the leading causes of loss in crop productivity. Plants respond to Pi deficiency by increasing Pi acquisition and remobilization involving organic and inorganic Pi transporters. Here, we report the functional characterization of a putative organic Pi transporter, Glycerol-3-phosphate permease (G3Pp) family, comprising five members (AtG3Pp1 to -5) in Arabidopsis (Arabidopsis thaliana). AtG3Pp1 and AtG3Pp2 showed 24-and 3-fold induction, respectively, in the roots of Pi-deprived seedlings, whereas Pi deficiency-mediated induction of AtG3Pp3 and -4 was evident in both roots and shoots. Furthermore, promoter-ß-glucuronidase (GUS) fusion transgenics were generated for AtG3Pp2 to -5 for elucidation of their in planta role in Pi homeostasis. During Pi starvation, there was a strong expression of the reporter gene driven by AtG3Pp4 promoter in the roots, shoots, anthers, and siliques, whereas GUS expression was specific either to the roots (AtG3Pp3) or to stamens and siliques (AtG3Pp5) in other promoter-GUS fusion transgenics. Quantification of reporter gene activities further substantiated differential responses of AtG3Pp family members to Pi deprivation. A distinct pattern of reporter gene expression exhibited by AtG3Pp3 and AtG3Pp5 during early stages of germination also substantiated their potential roles during seedling ontogeny. Furthermore, an AtG3Pp4 knockdown mutant exhibited accentuated total lateral root lengths under +phosphorus and -phosphorus conditions compared with the wild type. Several Pi starvation-induced genes involved in root development and/or Pi homeostasis were up-regulated in the mutant. A 9-fold induction of AtG3Pp3 in the mutant provided some evidence for a lack of functional redundancy in the gene family. These results thus reflect differential roles of members of the G3Pp family in the maintenance of Pi homeostasis.

Genetic and genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation in Arabidopsis.

Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes to cope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated an Arabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almost all aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused by overexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) in both its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-type plants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pi starvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants can be induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genes include several important Pi signaling components and those that are directly involved in Pi transport, mobilization, and distribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other both synergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compelling evidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate the signaling mechanism that controls plant responses to this particular nutritional stress.

Arabidopsis Pht1;5 mobilizes phosphate between source and sink organs and influences the interaction between phosphate homeostasis and ethylene signaling.

Phosphorus (P) remobilization in plants is required for continuous growth and development. The Arabidopsis (Arabidopsis thaliana) inorganic phosphate (Pi) transporter Pht1;5 has been implicated in mobilizing stored Pi out of older leaves. In this study, we used a reverse genetics approach to study the role of Pht1;5 in Pi homeostasis. Under low-Pi conditions, Pht1;5 loss of function (pht1;5-1) resulted in reduced P allocation to shoots and elevated transcript levels for several Pi starvation-response genes. Under Pi-replete conditions, pht1;5-1 had higher shoot P content compared with the wild type but had reduced P content in roots. Constitutive overexpression of Pht1;5 had the opposite effect on P distribution: namely, lower P levels in shoots compared with the wild type but higher P content in roots. Pht1;5 overexpression also resulted in altered Pi remobilization, as evidenced by a greater than 2-fold increase in the accumulation of Pi in siliques, premature senescence, and an increase in transcript levels of genes involved in Pi scavenging. Furthermore, Pht1;5 overexpressors exhibited increased root hair formation and reduced primary root growth that could be rescued by the application of silver nitrate (ethylene perception inhibitor) or aminoethoxyvinylglycine (ethylene biosynthesis inhibitor), respectively. Together, these data indicate that Pht1;5 plays a critical role in mobilizing Pi from P source to sink organs in accordance with developmental cues and P status. The study also provides evidence for a link between Pi and ethylene signaling pathways.

ATL8, a RING E3 ligase, modulates root growth and phosphate homeostasis in Arabidopsis.

Ubiquitination-mediated post-translational modification of proteins is a pivotal regulatory mechanism involved in the growth and development of the plant. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of RING-type ubiquitin ligases (E3) and ATL8 is a membrane-localized protein. Here, a reverse genetics approach was used to elucidate the role of ATL8 in phosphate (Pi) homeostasis. Deficiencies of Pi and sucrose (Suc) enhanced the relative expression level of ATL8 in different tissues of the wild-type (Wt). The relative expression level of ATL8 was attenuated and augmented in the mutant (atl8) and overexpression lines (Oe1 and Oe2), respectively. There were significant reductions in different root traits, root hairs, root to shoot ratio, and total Pi content in atl8 compared with the Wt under different Pi regimes. On the contrary, Oe1 and Oe2 lines exhibited enhancement in some of these traits. Noticeably, anthocyanin content was significantly reduced in Oe1 and Oe2 compared with the Wt and atl8 under P- condition. Abscisic acid (ABA) treatment led to an increase in the primary root length of atl8 compared with the Wt, suggesting a cross-talk between ABA and ATL8 on root growth. Furthermore, the relative expression levels of the genes involved in the maintenance of Pi homeostasis (WRKY75, RNS1, E3L, and ACP5) were differentially modulated in atl8, Oe1, and Oe2 compared with the Wt under different Pi regimes. The results revealed the pivotal role of ATL8 in mediating morphophysiological and molecular adaptive responses to Pi deficiency.

Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis.

• With the exception of root hair development, the role of the phytohormone ethylene is not clear in other aspects of plant responses to inorganic phosphate (Pi) starvation. • The induction of AtPT2 was used as a marker to find novel signalling components involved in plant responses to Pi starvation. Using genetic and chemical approaches, we examined the role of ethylene in the regulation of plant responses to Pi starvation. • hps2, an Arabidopsis mutant with enhanced sensitivity to Pi starvation, was identified and found to be a new allele of CTR1 that is a key negative regulator of ethylene responses. 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, increases plant sensitivity to Pi starvation, whereas the ethylene perception inhibitor Ag+ suppresses this response. The Pi starvation-induced gene expression and acid phosphatase activity are also enhanced in the hps2 mutant, but suppressed in the ethylene-insensitive mutant ein2-5. By contrast, we found that ethylene signalling plays a negative role in Pi starvation-induced anthocyanin production. • These findings extend the roles of ethylene in the regulation of plant responses to Pi starvation and will help us to gain a better understanding of the molecular mechanism underlying these responses.

Histone H2A.Z regulates the expression of several classes of phosphate starvation response genes but not as a transcriptional activator.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Here, we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi starvation response (PSR) genes in Arabidopsis (Arabidopsis thaliana). The loss of H2A.Z at these target loci results in their derepression in arp6 mutants and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.

Molecular identification and characterization of a gene associated with the onset of tapping panel dryness (TPD) syndrome in rubber tree (Hevea brasiliensis Muell.) by mRNA differential display.

In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.

Promoter deletion analysis elucidates the role of cis elements and 5'UTR intron in spatiotemporal regulation of AtPht1;4 expression in Arabidopsis.

The high-affinity phosphate transporter AtPht1;4 (Arabidopsis phosphate transporter1;4) is not only induced in response to inorganic phosphate (Pi) starvation but also preferentially expressed in the roots of Arabidopsis. In this study, we carried out AtPht1;4 promoter deletion analysis to identify regions that control the Pi responsiveness and spatiotemporal expression of the gene. Expression cassettes with truncated promoter fragments cloned to GUS (beta-glucuronidase) coding sequence were developed. Full-length promoter (-2327) and truncations up to -1436 (from the translational start) showed normal expression of GUS in various parts of the plants. The Pi responsiveness and inducibility of the reporter gene remained unaltered. However, deletion of the promoter region containing the first PHR1-binding site (P1BS) motif (-1350) abolished the AtPht1;4 expression in roots but not in aerial parts. A 164-bp region immediately upstream of the transcription start site appears to be sufficient for the basal expression of the gene. Interestingly, the 5'UTR (5' untranslated region) intron exhibited weak promoter activity as evidenced by its ability to drive the expression of AtPht1;4 in stipules and reproductive organs. Further analyses showed that the 5'UTR intron is essential for AtPht1;4 expression in root tips besides enhancing the level of expression in roots during Pi starvation. However, expression of AtPht1;4 in aerial parts of the plant was not influenced by the intron. Together these results suggest that expression of AtPht1;4 in the roots and aerial parts is regulated by independent mechanisms.

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

Arabidopsis thaliana mutant lpsi reveals impairment in the root responses to local phosphate availability.

Phosphate (Pi) deficiency triggers local Pi sensing-mediated inhibition of primary root growth and development of root hairs in Arabidopsis (Arabidopsis thaliana). Generation of activation-tagged T-DNA insertion pools of Arabidopsis expressing the luciferase gene (LUC) under high-affinity Pi transporter (Pht1;4) promoter, is an efficient approach for inducing genetic variations that are amenable for visual screening of aberrations in Pi deficiency responses. Putative mutants showing altered LUC expression during Pi deficiency were identified and screened for impairment in local Pi deficiency-mediated inhibition of primary root growth. An isolated mutant was analyzed for growth response, effects of Pi deprivation on Pi content, primary root growth, root hair development, and relative expression levels of Pi starvation-responsive (PSR) genes, and those implicated in starch metabolism and Fe and Zn homeostasis. Pi deprived local phosphate sensing impaired (lpsi) mutant showed impaired primary root growth and attenuated root hair development. Although relative expression levels of PSR genes were comparable, there were significant increases in relative expression levels of IRT1, BAM3 and BAM5 in Pi deprived roots of lpsi compared to those of the wild-type. Better understanding of molecular responses of plants to Pi deficiency or excess will help to develop suitable remediation strategies for soils with excess Pi, which has become an environmental concern. Hence, lpsi mutant will serve as a valuable tool in identifying molecular mechanisms governing adaptation of plants to Pi deficiency.

Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis.

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.

Molecular cloning and characterization of phosphate (Pi) responsive genes in Gulf ryegrass (Lolium multiflorum L.): a Pi hyperaccumulator.

Gulf annual ryegrass has been identified as potential Pi hyperaccumulator, however the molecular mechanism remains largely unknown. A suppression subtractive hybridization (SSH) analysis was used to evaluate the phosphate (Pi) responsive genome expression pattern changes in Gulf annual ryegrass (Lolium multiflorum L.). Differential screening identified 384 putative Pi-starvation induced cDNAs. Bioinformatic analysis revealed that 116 cDNAs are nonredundant unigenes of which 108 exhibited high similarities with Genbank entries. The differential expression patterns of 13 cDNAs, representing diverse functional categories, were confirmed by RNA gel blot analysis. Further, detailed molecular analysis of three genes (LmPAP1, LmIPS1 and LmIDS1) was carried out by cloning and characterization of full-length cDNAs. LmPAP1 is 1,414 bp in length with an open reading frame (ORF) of 1,188 bp capable of encoding an N-terminal signal peptide of 26 amino acids. LmIPS1 gene is a member of TPSI1/Mt4 family that contains 3 short ORFs. The cDNA of LmIDS1 is 346 bp in size including a single ORF of 222 nucleotides that encodes 74 amino acid proteins, exhibiting homology with IDS1 with similarity to type 2 metallothionein like protein. In our preliminary screening of different genotypes of annual ryegrass for hyperaccumulation of Pi in their shoots, Gulf and Urugrary showed significant differences with values of 1.0% and 0.7%, respectively. Since it is logical to assume a plausible correlation that may exist between Pi-accumulation in the shoots and the expression of Pi-responsive genes, the expression of LmPAP1, LmIPS1 and LmIDS1 was evaluated in these two genotypes grown under different Pi regimes. Although there was a significant induction of these genes in both the genotypes grown under Pi-deprived condition, the abundance of LmPAP1 transcripts was relatively higher in the Gulf genotype as compared to that in the Urugrary genotype. A similar trend was observed in qRT-PCR data of other tested genotypes of annual ryegrasses. This suggests the potential role of LmPAP1 in accumulation of Pi in Gulf grass. In addition, Gulf grass genotype revealed higher levels of total P, (33)Pi uptake, and APase activity as compared to Urugrary. Together, these results suggest that the Gulf ryegrass has evolved mechanisms to acquire and hyperaccumulate more Pi under different Pi regimes by activating multiple Pi acquisition and mobilization mechanisms.

Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis.

The limited availability of phosphate (Pi) in most soils results in the manifestation of Pi starvation responses in plants. To dissect the transcriptional regulation of Pi stress-response mechanisms, we have characterized the biological role of MYB62, an R2R3-type MYB transcription factor that is induced in response to Pi deficiency. The induction of MYB62 is a specific response in the leaves during Pi deprivation. The MYB62 protein localizes to the nucleus. The overexpression of MYB62 resulted in altered root architecture, Pi uptake, and acid phosphatase activity, leading to decreased total Pi content in the shoots. The expression of several Pi starvation-induced (PSI) genes was also suppressed in the MYB62 overexpressing plants. Overexpression of MYB62 resulted in a characteristic gibberellic acid (GA)-deficient phenotype that could be partially reversed by exogenous application of GA. In addition, the expression of SOC1 and SUPERMAN, molecular regulators of flowering, was suppressed in the MYB62 overexpressing plants. Interestingly, the expression of these genes was also reduced during Pi deprivation in wild-type plants, suggesting a role for GA biosynthetic and floral regulatory genes in Pi starvation responses. Thus, this study highlights the role of MYB62 in the regulation of phosphate starvation responses via changes in GA metabolism and signaling. Such cross-talk between Pi homeostasis and GA might have broader implications on flowering, root development and adaptive mechanisms during nutrient stress.

Variations in the composition of gelling agents affect morphophysiological and molecular responses to deficiencies of phosphate and other nutrients.

Low inorganic phosphate (Pi) availability triggers an array of spatiotemporal adaptive responses in Arabidopsis (Arabidopsis thaliana). There are several reports on the effects of Pi deprivation on the root system that have been attributed to different growth conditions and/or inherent genetic variability. Here we show that the gelling agents, largely treated as inert components, significantly affect morphophysiological and molecular responses of the seedlings to deficiencies of Pi and other nutrients. Inductively coupled plasma-mass spectroscopy analysis revealed variable levels of elemental contaminants not only in different types of agar but also in different batches of the same agar. Fluctuating levels of phosphorus (P) in different agar types affected the growth of the seedlings under Pi-deprivation condition. Since P interacts with other elements such as iron, potassium, and sulfur, contaminating effects of these elements in different agars were also evident in the Pi-deficiency-induced morphological and molecular responses. P by itself acted as a contaminant when studying the responses of Arabidopsis to micronutrient (iron and zinc) deficiencies. Together, these results highlighted the likelihood of erroneous interpretations that could be easily drawn from nutrition studies when different agars have been used. As an alternative, we demonstrate the efficacy of a sterile and contamination-free hydroponic system for dissecting morphophysiological and molecular responses of Arabidopsis to different nutrient deficiencies.

The phosphate transporter PHT4;6 is a determinant of salt tolerance that is localized to the Golgi apparatus of Arabidopsis.

Insertion mutations that disrupt the function of PHT4;6 (At5g44370) cause NaCl hypersensitivity of Arabidopsis seedlings that is characterized by reduced growth of the primary root, enhanced lateral branching, and swelling of root tips. Mutant phenotypes were exacerbated by sucrose, but not by equiosmolar concentrations of mannitol, and attenuated by low inorganic phosphate in the medium. Protein PHT4;6 belongs to the Major Facilitator Superfamily of permeases that shares significant sequence similarity to mammalian type-I Pi transporters and vesicular glutamate transporters, and is a member of the PHT4 family of putative intracellular phosphate transporters of plants. PHT4;6 localizes to the Golgi membrane and transport studies indicate that PHT4;6 facilitates the selective transport of Pi but not of chloride or inorganic anions. Phenotypic similarities with other mutants displaying root swelling suggest that PHT4;6 likely functions in protein N-glycosylation and cell wall biosynthesis, which are essential for salt tolerance. Together, our results indicate that PHT4;6 transports Pi out of the Golgi lumenal space for the re-cycling of the Pi released from glycosylation processes.

Biochemical and molecular analysis of LePS2;1: a phosphate starvation induced protein phosphatase gene from tomato.

Adaptation of plants to phosphate (Pi) deficiency is a complex process involving host of biochemical changes. These changes are integrated at transcriptional level by Pi starvation mediated signal transduction pathway. Many of the signaling processes are regulated by reversible protein phosphorylation directed by protein kinases and protein phosphatases. In this study, we report the characterization of a protein phosphatase gene (LePS2;1) from tomato induced during phosphate starvation. The bacterially expressed recombinant LePS2;1 protein readily dephosphorylated a synthetic phospho-Ser/Thr peptide. Okadaic acid, an inhibitor of Ser/Thr protein phosphatases, suppressed the enzyme activity. Western blot analysis revealed the Pi starvation dependent accumulation of LePS2;1 protein. Over-expression of LePS2;1 in tomato plants resulted in increased anthocyanin accumulation and acid phosphatase activity under Pi sufficient condition. Transgenic plants exhibited distinct changes in morphology and delayed flower initiation. These results provide evidence that the protein phosphatase LePS2;1, plays an important role in phosphate starvation induced processes in tomato. To our knowledge this is the first comprehensive analysis of a protein phosphatase induced during phosphate starvation.