Yi-Fang Chen

The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in Arabidopsis

Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and β-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression.

Arabidopsis WRKY45 Transcription Factor Activates PHOSPHATE TRANSPORTER1;1 Expression in Response to Phosphate Starvation

Overexpression of a transcription factor enhances Arabidopsis phosphate uptake by activating transporter expression. The WRKY transcription factor family has more than 70 members in the Arabidopsis (Arabidopsis thaliana) genome, and some of them are involved in plant responses to biotic and abiotic stresses. This study evaluated the role of WRKY45 in regulating phosphate (Pi) uptake in Arabidopsis. WRKY45 was localized in the nucleus and mainly expressed in roots. During Pi starvation, WRKY45 expression was markedly induced, typically in roots. WRKY45 overexpression in Arabidopsis increased Pi content and uptake, while RNA interference suppression of WRKY45 decreased Pi content and uptake. Furthermore, the WRKY45-overexpressing lines were more sensitive to arsenate, the analog of Pi, compared with wild-type seedlings. These results indicate that WRKY45 positively regulates Arabidopsis Pi uptake. Quantitative real-time polymerase chain reaction and β-glucuronidase staining assays showed that PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression was enhanced in the WRKY45-overexpressing lines and slightly repressed in the WRKY45 RNA interference line. Chromatin immunoprecipitation and eclectrophoretic mobility shift assay results indicated that WRKY45 can bind to two W-boxes within the PHT1;1 promoter, confirming the role of WRKY45 in directly up-regulating PHT1;1 expression. The pht1;1 mutant showed decreased Pi content and uptake, and overexpression of PHT1;1 resulted in enhanced Pi content and uptake. Furthermore, the PHT1;1-overexpressing line was much more sensitive to arsenate than WRKY45-overexpressing and wild-type seedlings, indicating that PHT1;1 overexpression can enhance Arabidopsis Pi uptake. Moreover, the enhanced Pi uptake and the increased arsenate sensitivity of the WRKY45-overexpressing line was impaired by pht1;1 (35S:WRKY45-18::pht1;1), demonstrating an epistatic genetic regulation between WRKY45 and PHT1;1. Together, our results demonstrate that WRKY45 is involved in Arabidopsis response to Pi starvation by direct up-regulation of PHT1;1 expression.

Membrane transporters for nitrogen, phosphate and potassium uptake in plants.

Nitrogen, phosphorous and potassium are essential nutrients for plant growth and development. However, their contents in soils are limited so that crop production needs to invest a lot for fertilizer supply. To explore the genetic potentialities of crops (or plants) for their nutrient utilization efficiency has been an important research task for many years. In fact, a number of evidences have revealed that plants, during their evolution, have developed many morphological, physiological, biochemical and molecular adaptation mechanisms for acquiring nitrate, phosphate and potassium under stress conditions. Recent discoveries of many transporters and channels for nitrate, phosphate and potassium uptake have opened up opportunities to study the molecular regulatory mechanisms for acquisition of these nutrients. This review aims to briefly discuss the genes and gene families for these transporters and channels. In addition, the functions and regulation of some important transporters and channels are particularly emphasized.

WRKY42 Modulates Phosphate Homeostasis through Regulating Phosphate Translocation and Acquisition in Arabidopsis

An Arabidopsis transcription factor modulates phosphate homeostasis by regulating phosphate uptake and translocation. The Arabidopsis (Arabidopsis thaliana) WRKY transcription factor family has more than 70 members, and some of them have been reported to play important roles in plant response to biotic and abiotic stresses. This study shows that WRKY42 regulated phosphate homeostasis in Arabidopsis. The WRKY42-overexpressing lines, similar to the phosphate1 (pho1) mutant, were more sensitive to low-inorganic phosphate (Pi) stress and had lower shoot Pi content compared with wild-type plants. The PHO1 expression was repressed in WRKY42-overexpressing lines and enhanced in the wrky42 wrky6 double mutant. The WRKY42 protein bound to the PHO1 promoter under Pi-sufficient condition, and this binding was abrogated during Pi starvation. These data indicate that WRKY42 modulated Pi translocation by regulating PHO1 expression. Furthermore, overexpression of WRKY42 increased root Pi content and Pi uptake, whereas the wrky42 mutant had lower root Pi content and Pi uptake rate compared with wild-type plants. Under Pi-sufficient condition, WRKY42 positively regulated PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression by binding to the PHT1;1 promoter, and this binding was abolished by low-Pi stress. During Pi starvation, the WRKY42 protein was degraded through the 26S proteasome pathway. Our results showed that AtWRKY42 modulated Pi homeostasis by regulating the expression of PHO1 and PHT1;1 to adapt to environmental changes in Pi availability.

Abscisic Acid Modulates Seed Germination via ABA INSENSITIVE5-Mediated PHOSPHATE1

Arabidopsis PHO1 modulates ABA-mediated seed germination and is a key target of transcription factor ABI5. The phytohormone abscisic acid (ABA) controls many developmental and physiological processes. Here, we report that PHOSPHATE1 (PHO1) participates in ABA-mediated seed germination and early seedling development. The transcription of PHO1 was obviously enhanced during seed germination and early seedling development and repressed by exogenous ABA. The pho1 mutants (pho1-2, pho1-4, and pho1-5) showed ABA-hypersensitive phenotypes, whereas the PHO1-overexpressing lines were ABA-insensitive during seed germination and early seedling development. The expression of PHO1 was repressed in the ABI5-overexpressing line and elevated in the abi5 mutant, and ABI5 can bind to the PHO1 promoter in vitro and in vivo, indicating that ABI5 directly down-regulated PHO1 expression. Disruption of PHO1 abolished the ABA-insensitive germination phenotypes of abi5 mutant, demonstrating that PHO1 was epistatic to ABI5. Together, these data demonstrate that PHO1 is involved in ABA-mediated seed germination and early seedling development and transcriptionally regulated by ABI5.

The Ubiquitin E3 Ligase PRU1 Regulates WRKY6 Degradation to Modulate Phosphate Homeostasis in Response to Low-Pi Stress in Arabidopsis

Arabidopsis PRU1 is an E3 ubiquitin ligase that modulates phosphate translocation from roots to shoots by directly regulating the degradation of WRKY6 under low-Pi stress. Since phosphorus is an essential nutrient for plants, plants have evolved a number of adaptive mechanisms to respond to changes in phosphate (Pi) supply. Previously, we reported that the transcription factor WRKY6 modulates Pi homeostasis by downregulating PHOSPHATE1 (PHO1) expression and that WRKY6 is degraded during Pi starvation in Arabidopsis thaliana. However, the molecular mechanism underlying low-Pi-induced WRKY6 degradation was unknown. Here, we report that a ubiquitin E3 ligase, PHOSPHATE RESPONSE UBIQUITIN E3 LIGASE1 (PRU1), modulates WRKY6 protein levels in response to low-Pi stress. A pru1 mutant was more sensitive than the wild type to Pi-deficient conditions, exhibiting a reduced Pi contents in the shoot, similar to the pho1-2 mutant and WRKY6-overexpressing line. PRU1 interacted with WRKY6 in vitro and in vivo. Under low-Pi stress, the ubiquitination and subsequent degradation of WRKY6, as well as the consequential enhancement of PHO1 expression, were impaired in pru1. PRU1 complementation lines displayed no obvious differences compared with wild-type plants. Further genetic analysis showed that disruption of WRKY6 abolished the low-Pi sensitivity of pru1, indicating that WRKY6 functioned downstream of PRU1. Taken together, this study uncovers a mechanism by which PRU1 modulates Pi homeostasis, through regulating the abundance of WRKY6 in response to low-Pi stress in Arabidopsis.