Negative regulation of phosphate starvation-induced genes

Abstract

Phosphate (Pi) deficiency is a major nutritional problem faced by plants in many agro-ecosystems. This deficiency results in altered gene expression leading to physiological and morphological changes in plants. Altered gene expression is presumed to be due to interaction of regulatory sequences (cis-elements) present in the promoters with DNA binding factors (trans-factors). In this study, we analyzed the expression and DNA-protein interaction of promoter regions of Pi starvation-induced genes AtPT2 and TPSI1. AtPT2 encodes the high-affinity Pi transporter in Arabidopsis, whereas TPSI1 codes for a novel gene induced in the Pi-starved tomato (Lycopersicon esculentum). Expression of AtPT2 was induced rapidly under Pi deficiency and increased with decreasing concentrations of Pi. Abiotic stresses except Pi starvation had no affect on the expression of TPSI1. DNA mobility-shift assays indicated that specific sequences of AtPT2 and TPSI1 promoter interact with nuclear protein factors. Two regions of AtPT2 and TPSI1 promoters specifically bound nuclear protein factors from Pi-sufficient plants. Interestingly, the DNA binding activity disappeared during Pi starvation, leading to the hypothesis that Pi starvation-induced genes may be under negative regulation.

Figure 1

Primer extension analysis transcription start sites of AtPT2 and TPSI1. The transcription start site was mapped to 172 and 91 bp upstream of the translational start site for AtPT2 and TPSI1, respectively. The total RNA isolated from Pi-sufficient (P+) and -deficient (P−) seedlings were used for mapping transcription start site of AtPT2. The poly(A⁺) RNA (a) and total RNA (b) isolated from Pi-starved roots of tomato were used to map TPSI1 transcriptional start site. DNA sequencing ladders were used to determine the size of the primer extended products.

Figure 2

Nucleotide sequence of promoter region of the AtPT2 gene. The putative transcription start site indicated by the bold letter is numbered +1. The promoter fragments interacting with nuclear factors are shown in bold. The underlined palindromic sequence ATGCCAT represents the conserved motif. The sequences showing similarity to the NIT-2 binding domain found in the regions of the promoter interacting with proteins are shaded.

Figure 3

TPSI1 is specifically induced under Pi starvation. Hydroponically grown tomato plants were exposed to 4°C (Cold), 37°C (Heat), desiccation (Des.), salt stress (Salt), and Pi starvation (P−) for 4 d. RNA extracted from leaves and roots was subjected to northern-blot analysis with ³²P-labeled TPSI1 cDNA fragment as the probe. The ethidium bromide stained gel below shows uniform loading of RNA samples.

Figure 4

Concentration-dependent and temporal expression of Pi transporters. A, Arabidopsis plants grown in liquid culture for 7 d were transferred to medium containing indicated amounts (μm) of Pi. Five days after the treatment, plants were harvested and stored at −70°C. Total RNA extracted from the plants was subjected to northern analysis with AtPT1 and AtPT2 cDNAs as probes. The ethidium bromide-stained gel below shows uniform loading of RNA samples. B, Seven-day-old Arabidopsis plants grown in liquid culture were subject to Pi starvation for indicated time. Total RNA was extracted and subjected to northern analysis with labeled AtPT1 and AtPT2 cDNAs as probes

Figure 5

Nuclear protein factors are not associated with promoters during Pi starvation. A, Map of the different promoter fragments of AtPT2 used in gel-shift assays. Dotted lines indicate fragments that interacted with protein factors. The location of an intron and the transcriptional start site (1+) are also shown. B, Labeled upstream fragments (1–6) of the promoter of AtPT2 were incubated with nuclear protein extracts (10 μg) obtained from Pi-sufficient (+) and -deficient (−) Arabidopsis plants. The reaction mix was analyzed on a polyacrylamide gel. C, Map of the different promoter fragments of TPSI1 used in gel-shift assays. Bold lines indicate the fragments interacting with protein factors. The transcriptional start site is marked as +1. D, Labeled upstream fragments (I–VI) were mixed with nuclear extracts (10 μg) isolated from Pi-sufficient (+) and -deficient (−) tomato roots. The reaction mix was analyzed on a polyacrylamide gel. E, Fragment III of the TPSI1 promoter interacted with leaf proteins extracted from Pi-sufficient plants, whereas the other fragments showed no interaction (data not shown).

Figure 6

Nuclear proteins interact specifically with promoter fragments. A, Labeled DNA fragment IV of TPSI1 promoter was mixed with nuclear extracts obtained from Pi-starved (−) or -sufficient (+) Arabidopsis tissue. Indicated mass excess of unlabeled fragment IV was added as specific competitors. Increasing concentrations of the competitor progressively reduced the binding of the labeled fragment. B, Increasing quantities of pBluescript DNA digested with HhaI and Sau3A were added to the DNA (TPSI1 promoter fragment IV)-protein interaction mix. Lack of competition with increasing mass excess of non-specific competitor indicated that the interaction between the DNA and the nuclear proteins is specific. C, The DNA binding factors are proteinaceous in nature. Nuclear proteins from Pi-sufficient tomato roots were subject to RNaseI and ProteinaseK (PK) treatments and heat denaturation (65°C and 100°C) for 10 min. The treated nuclear proteins were used in gel retardation reactions with the TPSI1 promoter fragment IV. The treatments that denature proteins resulted in a loss of DNA-protein interaction, whereas RNaseI treatment did not affect binding.