Phosphate is an essential but limited macronutrient for all plants. In response to limited levels of phosphate, plants have developed highly specialized developmental, biochemical, and molecular responses. To further expand our knowledge of the phosphate starvation induced signal transduction pathway in plants, the expression of the phosphate starvation inducible Purple Acid Phosphatase 1 (PAP1) gene was studied in transgenic Arabidopsis. While few components have been identified regulating gene expression under phosphate starvation conditions in plants, one cis regulatory element recognized by the MYB transcriptions factor Phosphate Starvation Response 1 (PHR1) has been identified in many phosphate starvation induced (PSI) genes. PAP1 and many other genes examined during the course of the mutant characterization contain this cis element. Using the GUS reporter gene under control of the PAP1 promoter, a mutant screen was devised for plants showing abnormal PAP1 response to phosphate nutrition. Three mutant lines were identified and subsequently characterized for the phosphate starvation-induced signal-transduction pathway in Arabidopsis. Two mutants, BT1 and BT2, both with dominant mutations, showed increased GUS staining. The mutations in BT1 and BT2 are tightly linked to the transgene and to each other, but complementation analysis suggested that they are in different genes. Characterization of these mutants indicated that the PSI genes PAP1 and At4 (in BT1 roots), and RNS1 (in BT2 leaves) have alternative or additional methods of regulation other than PHR, even though these genes all contain PHR1 binding sites. A third mutant, BT3, had a phenotype similar to the PAP1 null-mutant and did not show PAP1 phosphatase activity under normal soil-grown conditions. Characterization of BT3 indicates that PAP1, RNS1, and AtIPS1 are not exclusively regulated by PHR1. In an attempt to map the BT3 mutant in a Columbia background by crossing with Landsberg erecta (Ler), it was discovered that the Ler ecotype does not show PAP1 phosphatase activity under normal soil-grown conditions. The PAP1 phosphatase regulatory trait, named BT5, was mapped to a 15,562 bp-region area containing only two genes between the GPA1 and ER markers on Chromosome 2.

Phosphorus is an essential element for plant growth and development. Due to its low availability, solubility and mobility, phosphate is often the limiting macronutrient for crops and other plants. Plants have evolved several responses to phosphate deficiency. However, very little is known about the molecular basis of these responses. Here, I study the expression of PAP12, its role in the phosphate starvation response and the interaction of its promoter with nuclear factors. Analysis of a PAP12 T-DNA insertion line (pap12-1) revealed PAP12 is responsible for the majority of the acid phosphatase activity detected by the standard in-gel assay. RNA gel blots showed that PAP12 was induced only by Pi deficiency, and not by general nutrient stress. PAP12 expression, at the RNA and protein level, reflected endogenous phosphate levels in two mutants with altered phosphate accumulation. In the pho1 mutant, PAP12 expression and activity were up-regulated with respect to wild-type plants, and in the pho2 mutant, PAP12 expression and activity were reduced. Analysis of the PAP12 promoter using promoter-GUS fusions revealed expression in leaves, roots, flowers, hydathodes, root tips, and pollen grains. This broad pattern of expression suggests that PAP12 functions throughout the plant in response to low phosphate concentrations. The results showed PAP12 does not play a major role in phosphate remobilization, acquisition or in helping plants cope with low phosphate environments. Instead, the major phenotype associated with PAP12 deficiency was a significant delay in flowering in the low-phosphate pho1 background and a slight acceleration of flowering in the high-phosphate pho2 background over-expressing PAP12. These results suggest that PAP12 may have a role in linking phosphate status with the transition to flowering. Finally, I used promoter deletion and DNA-protein interaction assay to understand PAP12 expression upon phosphate starvation. A 35-bp region of the PAP12 promoter was identified as an important phosphate regulatory cis-element required for induction during phosphate starvation. We isolated a 23.5 kDa nuclear factor, which binds to this 35-bp region of the PAP12 promoter in a phosphate-dependent manner. The work presented here will add to our knowledge about the molecular processes that regulate phosphate nutrition.

Phosphorus is an essential element for plant growth and development. Due to its low availability, solubility and mobility, phosphate is often the limiting macronutrient for crops and other plants. Plants have evolved several responses to phosphate deficiency. However, very little is known about the molecular basis of these responses. Here, I study the expression of PAP12, its role in the phosphate starvation response and the interaction of its promoter with nuclear factors. Analysis of a PAP12 T-DNA insertion line (pap12-1) revealed PAP12 is responsible for the majority of the acid phosphatase activity detected by the standard in-gel assay. RNA gel blots showed that PAP12 was induced only by Pi deficiency, and not by general nutrient stress. PAP12 expression, at the RNA and protein level, reflected endogenous phosphate levels in two mutants with altered phosphate accumulation. In the pho1 mutant, PAP12 expression and activity were up-regulated with respect to wild-type plants, and in the pho2 mutant, PAP12 expression and activity were reduced. Analysis of the PAP12 promoter using promoter-GUS fusions revealed expression in leaves, roots, flowers, hydathodes, root tips, and pollen grains. This broad pattern of expression suggests that PAP12 functions throughout the plant in response to low phosphate concentrations. The results showed PAP12 does not play a major role in phosphate remobilization, acquisition or in helping plants cope with low phosphate environments. Instead, the major phenotype associated with PAP12 deficiency was a significant delay in flowering in the low-phosphate pho1 background and a slight acceleration of flowering in the high-phosphate pho2 background over-expressing PAP12. These results suggest that PAP12 may have a role in linking phosphate status with the transition to flowering. Finally, I used promoter deletion and DNA-protein interaction assay to understand PAP12 expression upon phosphate starvation. A 35-bp region of the PAP12 promoter was identified as an important phosphate regulatory cis-element required for induction during phosphate starvation. We isolated a 23.5 kDa nuclear factor, which binds to this 35-bp region of the PAP12 promoter in a phosphate-dependent manner. The work presented here will add to our knowledge about the molecular processes that regulate phosphate nutrition.

Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of phosphate (Pi) from Pi monoesters and anhydrides within the acidic pH range. Induction of intracellular and secreted purple acid phosphatases (PAPs) is a widespread plant response to nutritional Pi-deficiency. The probable function of intracellular APases is to recycle Pi from expendable intracellular organophosphate pools, whereas secreted APases likely scavenge Pi from the organically bound Pi that is prevalent in most soils. Although the catalytic function and regulation of plant PAPs have been described, their physiological function in plants has not been fully established. Recent biochemical and proteomic studies indicated that AtPAP26 is the predominant intracellular (vacuolar) and a major secreted purple APase isozyme upregulated by Pi-starved (-Pi) Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular, biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant. Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pi-sufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to Pi-recycling and scavenging in –Pi Arabidopsis. / Thesis (Master, Biology) -- Queen's University, 2009-09-01 09:46:39.302

Post-transcriptional gene silencing (PTGS) is a powerful mechanism that can be adapted to genetically modify crop plants. PTGS operates in many plant signaling pathways including those mediating stress responses. Given the small number of miRNAs known, research on the characterization of stress-related micro-RNA (miRNA) and their targets could provide the basis for engineering stress tolerant traits in crops. Indeed, several examples of miRNA mediated crop tolerance have been reported. In the research presented here, we aimed to analyze the role of small non-coding RNA (smRNA) pathways involved in plant stress. In particular, we focused on miRNA-mediated PTGS in phosphate (Pi) starvation. The analysis was split into two research projects. First, to identify potential miRNA targets we began by analyzing the response and recovery of coding and long non-coding RNAs (lncRNA) to Pi starvation in shoot and root. The results obtained were the first genome-wide description of the root’s Pi starvation response and recovery. We found that the root's response involved a widely different set of genes than that of the shoot. In the second research project, the results of the first project were correlated with the responses of miRNA and trans-acting small-interfering RNA (tasiRNA) during Pi starvation. Many miRNA circuits have been predicted before, however, tasiRNA circuits are not as well defined. Therefore, we made use of the double-stranded RNA-binding protein 4 (DRB4) smRNA libraries to enhance our prediction of tasiRNAs. Altogether, we provided evidence to support the following miRNA-mRNA pairs that may function in Pi starvation: IPS1:miR399:PHO2; miR399:RS4; miR399:NF-YA10; miR398:CSD1/2; miR2111:TPS11; miR164:NAC6; miR157:TMO7; miR157:PSB28; RPS2:miR169:IPS2; miR397:LAC2; TAS4:PAP1; NR1:PAP1; and Chr3_1967672:TMO7. In general, we found that non-miR399 related circuits were active only during the root’s recovery from Pi starvation. The functional roles of the genes targeted by these PTGS circuits suggested that the local root response to Pi starvation was influenced by the plant's systemic response pathways via PHR1-mediated PTGS. Finally, since many PTGS targets function to modulate concentrations of reactive oxygen species and sucrose, we hypothesized that the candidate PTGS circuits found in our research mediate a general stress recovery process by modulating metabolites involved in signaling pathways.

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Phosphate (Pi) deficiency is a global problem for food production. Plants have evolved complex mechanisms to adapt to low Pi. We focused on the initial aspects of adaptation to low Pi - perception and immediate-early responses to changes in external Pi. To examine whether a labile repressor controls expression of the high affinity Pi transporter, Pht1;1, we performed electrophoretic mobility shift assays (EMSA) but observed only weak protein-DNA binding activity using extracts from Arabidopsis suspension cultures or seedlings. The regulatory role of different regions in Pht1;1 promoter was dissected by promoter deletion analysis, using uidA as a reporter. We identified two domains important for regulation: sequences between -1898 bp and - 932 bp are important for induction of Pht1;1 in low Pi; the intron in the 5’UTR impacts Pht1;1 expression in the young part of both primary and lateral root apices. A complementary approach to identify repressors of Pi starvation responses was pursued: We identified ZAT18, a putative transcription factor, as a candidate repressor. ZAT18 contains an EAR motif, a repressor domain in plants; the expression of ZAT18 responds to Pi starvation. Using transgenic lines with promoter::ZAT18-VENUS constructs, we studied its expression, localization and abundance in different levels of Pi availability: ZAT18 is mainly expressed in the nucleus of Arabidopsis root hair cells. Its accumulation was induced by 4 day Pi starvation. We also performed a microarray analysis to examine global gene expression levels during Pi starvation and rapid recovery. Our data indicated that 258 genes were induced and 188 genes were suppressed during Pi starvation. For most of these genes, responses were reversed after 4 hour Pi recovery. Further study of these genes will help to define targets of the early Pi starvation-signalling pathway.

Phosphate (Pi) is an essential nutrient for plants, required for plant growth and seed viability. When Pi is limited, plants undergo dynamic morphological and metabolic changes to leverage available Pi, known as the Phosphate Starvation Response (PSR). The inositol phosphate (InsP) signaling pathway is a crucial element of the plant's ability to regulate the PSR and respond to changing energy conditions. InsPs are synthesized from the cyclic 6-carbon polyol scaffold, myo-inositol. Inositol hexakisphosphate (InsP6) is the most abundant InsP signaling molecule and can be phosphorylated by the multifunctional inositol tetrakisphosphate 1-kinase 1 (ITPK1) and diphosphoinositol pentakisphosphate (VIP) kinases, resulting in inositol pyrophosphates (PP-InsPs). PP-InsPs have high energy bonds and have been linked to Pi maintenance and energy homeostasis in yeast, plants, and mammals. However, the precise mechanism(s) by which PP-InsPs act within plant signaling pathways remains to be determined. Two approaches to understand the role of PP-InsPs in plants are described within this dissertation. The first approach analyzes genetic loss-of-function vip1/vip2 double mutants, and their responses to low Pi conditions. Specifically, vip1/vip2 double mutant gene expression and lipid remodeling patterns in response to low Pi were characterized. We found that vip1-2/vip2-2 had an impacted lipid remodeling response under low Pi conditions, whereas ipk1 had altered lipid composition under Pi-replete conditions. In a complementary approach, a gain-of-function in either the ITPK1 or the kinase domain of VIP (VIP2KD) were constructed in transgenic Arabidopsis thaliana plants. Both ITPK1 and VIP2KD transgenic plants contain elevated levels of the specific inositol pyrophosphate, InsP8. Elevated InsP8 in both types of plants results in changes in growth and senescence phenotypes, delayed time to flowering, Pi accumulation, and altered PSR gene expression. The data from both approaches suggest new roles for PP-InsPs in the regulation of the PSR and other signaling pathways in plants. To enhance my teaching and leadership skills, I participated in the Graduate Teaching Scholars (GTS) program. As a GTS, I worked with the Virginia Tech Research and Extension Experiential Learning (VT-REEL) program where I developed a structured mentorship program for undergraduate and graduate students and created a professional development workshop series. During the COVID-19 pandemic, I developed an online version of the VT-REEL program. Using inclusive pedagogy practices and surveys from the participants, we compiled the best practices for moving a summer undergraduate research program online. These practices come from surveyed participants in the 2020 and provides strategies that can be tailored to various online research experiences and be implemented in both online and in-person formats. / Doctor of Philosophy / Phosphate (Pi) is crucial for plant development and crop yield, but is often limited in soils. Pi-containing fertilizers are often added to supplement soils. Overuse of Pi-containing fertilizers can lead to Pi runoff and can devastate aquatic ecosystems. In addition, Pi is a limited, nonrenewable resource, with U.S. stores projected to be depleted in as little as 30 years. It is now crucial to develop crops that can feed a growing population with less Pi input. Here, we describe how changing levels of plant messenger molecules known as inositol pyrophosphates (PP-InsPs) impact the ability of plants to sense and respond to Pi. This knowledge advances understanding f how mineral nutrient physiology affects many plants traits, and can be harnessed to develop novel strategies to reduce Pi-application and overuse.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.