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Engineering Phosphorus Deficiency Report System And Analyzing Phosphate Redistribution Mechanism In Plants By Using Rice Phosphate Transporter Genes

Posted on:2015-06-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y T LiFull Text:PDF
GTID:1220330482970070Subject:Plant Nutrition
Abstract/Summary:PDF Full Text Request
Phosphorus (P) is a fundamental macronutrient required for plant growth and development. Although P is abundant in rhizospheres, bioavailable orthophosphate (Pi) for acquisition by plants is often present at extremely low level that affects plant growth and development and consequently crop productivity. Therefore to circumvent Pi limitation, soils are often amended with Pi-containing fertilizers. However the world’s reserves of easily mined rock Pi, as the major source of Pi fertilizers, are projected to be exhausted in near future. Meanwhile, over-application and very low utilization rate of Pi fertilizers causes eutrophication which is becoming a global environmental concern. Therefore, there is an urgent need to develop economically viable strategies for reducing overuse of Pi fertilizers by real-time monitoring of plant Pi status and elucidation of the mechanistic details governing acquisition, mobilization and its appropriate utilization for sustainable agriculture.Conventionally soluble and total Pi concentrations in plant tissues are quantified either by phosphomolybdate colorimetric assay or cost-prohibitive ICP-based spectroscopy techniques. Although advances in hyperspectral sensor technology provides a rapid way to assess plant nutrient status, narrow range of leaf optical property is a bottleneck in diagnosing real-time plant Pi status. Here, in this study a color-based visual reporter system was engineered that facilitated specific, accurate and rapid assessment of plant Pi status by both naked eyes and by spectral reflectance. Next, the focus of this study was to elucidate the molecular mechanism governing acquisition and mobilization of Pi. In rice (Oryza sativa), a few Pi transporters (PT) belonging to Pht1 (PHOSPHATE TRANSPORTER1) family have been shown to play pivotal roles in acquisition and/or mobilization of Pi under different Pi regime. However the role of OsPhtl;8 (OsPTS), one of the 13 members of Pht1 family, in the maintenance of Pi homeostasis has far from being elucidated. Therefore, to determine the role of OsPT8 in re-distribution of Pi inside the plant, transgenic rice was generated by specific knock-down of this gene expression in shoot and seeds by RNA interference. Highlights of the present study are listed below:1. Bioinformatic analysis of cis-elements on the promoters of Pi deficiency-induced rice PTs OsPht1;2 (OsPT2) and OsPhtl;6 (OsPT6) were performed to identify the one that would be ideal for generating transgenic rice that could readily indicate Pi status during growth under different Pi regime. Both OsPT2 and OsPT6 promoters have one P1BS and two W-box cis-elements. Based on the relative positions of P1BS and W-box in the promoters of these two genes, they were truncated into different lengths and subsequently fused with GUS reporter gene. The results clearly demonstrated that transgenic plants harboring reporter GUS gene fused with the promoter of either OsPT2 or OsPT6 containing only one P1BS element were faintly responsive to Pi starvation. In addition, W-box element(s) could possibly have multiple regulatory roles in altering Pi-starvation signaling in rice. The 2360-bp OsPT6 promoter, which showed strongest GUS activity in Pi depleted rice shoot, was further selected and evaluated in response to Pi starvation in heterologous tobacco plant. The results clearly showed that rice OsPT6 promoter could function specifically in inducing gene expression by P starvation in tobacco.2. For generating a visual Pi deficiency indictor plants, visible anthocyanin was selected, instead of GUS, as the reporter gene. MYB-related cauliflower transcription factor Pr, which activates a series of biosynthetic genes leading to the accumulation of colored anthocyanins, was first cloned and then generated OsPT6pro::Pr (PT6pro::Pr) construct for generating transgenic rice and tobacco. Under Pi-deprived condition, relative to wild-type, PT6pro::Pr transgenic tobacco leaves were dark purple due to abundant accumulation of anthocyanin pigment. RT-qPCR analysis revealed that the expression of external Pr gene under Pi starvation condition activated the endogenous anthocyanin biosynthetic pathway genes, resulting in abundant anthocyanins accumulation in tobacco. Although Pi deficiency triggered the expression of Pr in PT6pro::Pr transgenic rice, the visible purple pigment could not be detected in transgenic rice leaves. The observed difference could be attributed to substantial differences in anthocyanin biosynthetic pathway of dicot and monocot plants.3. To further evaluate the sensitivity and specificity of tobacco Pi deficiency reporter system for the diagnosis of Pi nutrition status in a temporal manner, transgenic plants were grown under different Pi regime, and also subjected to different abiotic stresses. With an increase in duration and severity of Pi deficiency, there was a concomitant increase in dark purple pigmentation in leaves of PT6pro::Pr transgenic tobacco and turned green rapidly upon replenishment with Pi. The results clearly demonstrated a high incidence of inverse correlation between Pi content and anthocyanin accumulation in transgenic tobacco leaves. Anthocyanin accumulation in transgenic tobacco was not detected when either deprived of other major-and micro-nutrients or subjected to salt, drought and cold stresses.4. Using hyperspectral sensing technology, Pi concentration in the leaves of transgenic tobacco could be predicted by the reflectance spectra at 554 nm wavelength with approximately 0.16 as the threshold value of Pi deficiency.5. OsPT8 was previously reported to play the broad role in Pi uptake and transport in rice, however, its role in re-distribution of Pi was not yet characterized. In this study, the expression of OsPT8 was knock-down selectively in rice shoot and/or seed endosperm by RNA-interference using RISBZ1 and GluB-1 promoters and designated these transgenic lines as SSRi and EnSRi, respectively. Since expression of OsPT8 in roots was not affected, the indirect effect of root Pi uptake on Pi-redistribution was avoided in the transgenic rice. In comparison to the stunted and nearly acarpous rice mutant ospt8 with constitutive silencing of OsPT8, the growth performance of both SSRi and EnSRi transgenic rice plants were relatively better. Their seed-setting rate and 1000-grain weight were decreased only by 10%-20% and 5%-22%, respectively, compared with those of the WT. The results clearly suggested that the stunted and acarpous phenotypes of ospt8 in previous report were due to attenuation of Pi acquisition by the mutants.6. To determine whether OsPT8 was involved in the remobilization of Pi from old to young blades, the hydroponic experiment was conducted to compare the alteration of both Pi and total P in roots, old blades and young blades across WT, SSRi and EnSRi mutants. Compared to WT and EnSRi lines, SSRi lines under Pi-deficient condition accumulated more P in old blades and less P in young blades. This differential accumulation of Pi correlated with attenuated and enriched transcripts of OsPT6 in old and young blades, respectively. These results strongly illustrate a pronounced role of OsPT8 in Pi redistribution from old leaves to young leaves during Pi starvation.7. To gain further insight into the potential role of OsPT8 in the maintenance of Pi homeostasis within rice seed, total P and phytic acids contents of embryo and endosperm were assayed. Relative to the WT, SSRi lines showed a substantial decrease of total P content in both endosperm (approximately 10%), and embryo (approximately 20-35%), while EnSRi lines showed about 50% decrease of total P content in embryos but similar P content in endosperm. These data thus support a novel role of OsPT8 in facilitating the allocation of Pi between embryo and endosperm. Interestingly, irrespective of P status of WT, SSRi and EnSRi lines, their phytic acid P content remained relatively stable. This suggested the priority of phytic acids homeostasis in Pi metabolism of the seeds. Overall, the results explicitly demonstrated that OsPT8 plays critical roles in redistribution of Pi from source to sink organs and also in the maintenance of Pi homeostasis in seeds.Taken these data together, the results demonstrated that plant phosphate transporter genes can be used not only in efficiently regulating Pi uptake and utilization, in particular in improving P-homeostasis as displayed by OsPT8, but also in engineering smart plant for specifically and real-time diagnosis of P-supply status in field.
Keywords/Search Tags:Pi transporter, P diagnosis, indactor plant, Pi redistribution
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