Athikkattuvalasu S. Karthikeyan

WRKY75 Transcription Factor Is a Modulator of Phosphate Acquisition and Root Development in Arabidopsis

Phosphate (Pi) deficiency limits plant growth and development, resulting in adaptive stress responses. Among the molecular determinants of Pi stress responses, transcription factors play a critical role in regulating adaptive mechanisms. WRKY75 is one of several transcription factors induced during Pi deprivation. In this study, we evaluated the role of the WRKY75 transcription factor in regulating Pi starvation responses in Arabidopsis (Arabidopsis thaliana). WRKY75 was found to be nuclear localized and induced differentially in the plant during Pi deficiency. Suppression of WRKY75 expression through RNAi silencing resulted in early accumulation of anthocyanin, indicating that the RNAi plants were more susceptible to Pi stress. Further analysis revealed that the expression of several genes involved in Pi starvation responses, including phosphatases, Mt4/TPS1-like genes, and high-affinity Pi transporters, was decreased when WRKY75 was suppressed. Consequently, Pi uptake of the mutant plant was also decreased during Pi starvation. In addition, when WRKY75 expression was suppressed, lateral root length and number, as well as root hair number, were significantly increased. However, changes in the root architecture were obvious under both Pi-sufficient and Pi-deficient conditions. This indicates that the regulatory effect of WRKY75 on root architecture could be independent of the Pi status of the plant. Together, these results suggest that WRKY75 is a modulator of Pi starvation responses as well as root development. WRKY75 is the first member of the WRKY transcription factor family reported to be involved in regulating a nutrient starvation response and root development.

Regulated expression of Arabidopsis phosphate transporters.

Phosphorus deficiency is one of the major abiotic stresses affecting plant growth. Plants respond to the persistent deficiency of phosphate (Pi) by coordinating the expression of genes involved in alleviation of the stress. The high-affinity Pi transporters are among the major molecular determinants that are activated during Pi stress. In this study, using three reporter genes (green fluorescent protein, luciferase, and β-glucuronidase) regulated by two Pi transporter promoters, we have carried out an extensive analysis of transcriptional and spatial regulation of gene expression. Activation of the genes was rapid, repressible, and specific in response to changes in Pi availability. The phytohormones auxin and cytokinin suppressed the expression of the reporter gene driven by the AtPT1promoter, and that of the native gene, suggesting that hormones may be involved in regulation of some component(s) of Pi starvation response pathway. These studies also provide molecular evidence for a potential role of high-affinity Pi transporters in mobilizing Pi into reproductive organs. The results suggest that members of the Pi transporter family may have similar but nonredundant functions in plants.

Differential Effects of Sucrose and Auxin on Localized Phosphate Deficiency-Induced Modulation of Different Traits of Root System Architecture in Arabidopsis

Phosphorus, one of the essential elements for plants, is often a limiting nutrient in soils. Low phosphate (Pi) availability induces sugar-dependent systemic expression of genes and modulates the root system architecture (RSA). Here, we present the differential effects of sucrose (Suc) and auxin on the Pi deficiency responses of the primary and lateral roots of Arabidopsis (Arabidopsis thaliana). Inhibition of primary root growth and loss of meristematic activity were evident in seedlings grown under Pi deficiency with or without Suc. Although auxin supplementation also inhibited primary root growth, loss of meristematic activity was observed specifically under Pi deficiency with or without Suc. The results suggested that Suc and auxin do not influence the mechanism involved in localized Pi sensing that regulates growth of the primary root and therefore delineates it from sugar-dependent systemic Pi starvation responses. However, the interaction between Pi and Suc was evident on the development of the lateral roots and root hairs in the seedlings grown under varying levels of Pi and Suc. Although the Pi+ Suc− condition suppressed lateral root development, induction of few laterals under the Pi− Suc− condition point to increased sensitivity of the roots to auxin during Pi deprivation. This was supported by expression analyses of DR5∷uidA, root basipetal transport assay of auxin, and RSA of the pgp19 mutant exhibiting reduced auxin transport. A significant increase in the number of lateral roots under the Pi− Suc− condition in the chalcone synthase mutant (tt4-2) indicated a potential role for flavonoids in auxin-mediated Pi deficiency-induced modulation of RSA. The study thus demonstrated differential roles of Suc and auxin in the developmental responses of ontogenetically distinct root traits during Pi deprivation. In addition, lack of cross talk between local and systemic Pi sensing as revealed by the seedlings grown under either the Pi− Suc− condition or in the heterogenous Pi environment highlighted the coexistence of Suc-independent and Suc-dependent regulatory mechanisms that constitute Pi starvation responses.

Phosphate starvation responses are mediated by sugar signaling in Arabidopsis

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be downstream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.

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.

Phosphite, an Analog of Phosphate, Suppresses the Coordinated Expression of Genes under Phosphate Starvation

Phosphate (Pi) and its analog phosphite (Phi) are acquired by plants via Pi transporters. Although the uptake and mobility of Phi and Pi are similar, there is no evidence suggesting that plants can utilize Phi as a sole source of phosphorus. Phi is also known to interfere with many of the Pi starvation responses in plants and yeast (Saccharomyces cerevisiae). In this study, effects of Phi on plant growth and coordinated expression of genes induced by Pi starvation were analyzed. Phi suppressed many of the Pi starvation responses that are commonly observed in plants. Enhanced root growth and root to shoot ratio, a hallmark of Pi stress response, was strongly inhibited by Phi. The negative effects of Phi were not obvious in plants supplemented with Pi. The expression of Pi starvation-induced genes such as LePT1, LePT2,AtPT1, and AtPT2 (high-affinity Pi transporters); LePS2 (a novel acid phosphatase);LePS3 and TPSI1 (novel genes); andPAP1 (purple acid phosphatase) was suppressed by Phi in plants and cell cultures. Expression of luciferase reporter gene driven by the Pi starvation-induced AtPT2 promoter was also suppressed by Phi. These analyses showed that suppression of Pi starvation-induced genes is an early response to addition of Phi. These data also provide evidence that Phi interferes with gene expression at the level of transcription. Synchronized suppression of multiple Pi starvation-induced genes by Phi points to its action on the early molecular events, probably signal transduction, in Pi starvation response.

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.

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.

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-β-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.

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.