Keke Yi

OsPTF1, a Novel Transcription Factor Involved in Tolerance to Phosphate Starvation in Rice

We report here on a novel transcription factor with a basic helix-loop-helix domain for tolerance to inorganic phosphate (Pi) starvation in rice (Oryza sativa). The gene is designated OsPTF1. The expression of OsPTF1 is Pi starvation induced in roots while constitutively expressed in shoots, as shown by northern-blot analysis. Overexpression of OsPTF1 enhanced tolerance to Pi starvation in transgenic rice. Tillering ability, root and shoot biomass, and phosphorus content of transgenic rice plants were about 30% higher than those of the wild-type plants in Pi-deficient conditions in hydroponic experiments. In soil pot and field experiments, more than 20% increase in tiller number, panicle weight, and phosphorus content was observed in transgenic plants compared to wild-type plants at low-Pi levels. In Pi-deficient conditions, transgenic rice plants showed significantly higher total root length and root surface area, which results in a higher instantaneous Pi uptake rate over their wild-type counterparts. Microarray analysis for transgenic plants overexpressing OsPTF1 has been performed to investigate the downstream regulation of OsPTF1.

A Rice Glutamate Receptor–Like Gene Is Critical for the Division and Survival of Individual Cells in the Root Apical Meristem

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor–like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor–like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor–like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.

Effects of genetic background and environment on QTLs and epistasis for rice (Oryza sativa L.) panicle number

Abstract A double-haploid (DH) population and a recombinant inbred (RI) line population, derived from a cross between a tropical japonica variety, Azucena, as male parent and two indica varieties, IR64 and IR1552, as female parents respectively, were used in both field and pot experiments for detecting QTLs and epistasis for rice panicle number in different genetic backgrounds and different environments. Panicle number (PN) was measured at maturity. A molecular map with 192 RFLP markers for the DH population and a molecular map with 104 AFLP markers and 103 RFLP markers for the RI population were constructed, in which 70 RFLP markers were the same. Six QTLs were identified in the DH population, including two detected from field experiments and four from pot experiments. The two QTLs, mapped on chromosomes 1 and 12, were identical in both field and pot experiments. In the RI population, nine QTLs were detected, five QTLs from field conditions and four from the pot experiments. Three of these QTLs were identical in both experimental conditions. Only one QTL, linked to CDO344 on chromosome 12, was detected across the populations and experiments. Different epistasitic interaction loci on PN were found under different populations and in different experimental conditions. One locus, flanked by RG323 and RZ801 on chromosome 1, had an additive effect in the DH population, but epistatic effects in the RI population. These results indicate that the effect of genetic background on QTLs is greater than that of environments, and epistasis is more sensitive to genetic background and environments than main-effect QTLs. QTL and epistatic loci could be interchangeable depending on the genetic backgrounds and probably on the environments where they are identified.

SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through Its Interaction with PHR2 in Rice

This study shows that rice SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2, a key transcription factor regulating phosphate signaling. SPX4 stabilization is dependent on phosphate concentration and appears to act as a regulatory point both for nuclear localization and for binding of PHR2 to P1BS cis-elements in target DNA. PHR2, a central regulator of phosphate signaling in rice, enhanced the expression of phosphate starvation-induced (PSI) genes and resulted in the enhancement of Pi acquisition under Pi deficiency stress. This occurred via PHR2 binding to a cis-element named the PHR1 binding sequences. However, the transcription level of PHR2 was not responsive to Pi starvation. So how is activity of transcription factor PHR2 adjusted to adapt diverse Pi status? Here, we identify an SPX family protein, Os-SPX4 (SPX4 hereafter), involving in Pi starvation signaling and acting as a negative regulator of PHR2. SPX4 is shown to be a fast turnover protein. When Pi is sufficient, through its interaction with PHR2, SPX4 inhibits the binding of PHR2 to its cis-element and reduces the targeting of PHR2 to the nucleus. However, when plants grow under Pi deficiency, the degradation of SPX4 is accelerated through the 26S proteasome pathway, thereby releasing PHR2 into the nucleus and activating the expression of PSI genes. Because the level of SPX4 is responsive to Pi concentration and SPX4 interacts with PHR2 and regulates its activity, this suggests that SPX4 senses the internal Pi concentration under diverse Pi conditions and regulates appropriate responses to maintain Pi homeostasis in plants.

cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit.

The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin (PIP2a), 9-cis-epoxycarotenoid dioxygenase (NCED1) and a negative regulator of gibberellin signal transduction (SPY); eight other genes participated in cell wall loosening or vesicle traffic.

Integrative Comparison of the Role of the PHOSPHATE RESPONSE1 Subfamily in Phosphate Signaling and Homeostasis in Rice

A subfamily of phosphate-responsive genes is functionally diverse in the regulation of phosphate signaling and homeostasis. Phosphorus (P), an essential macronutrient for all living cells, is indispensable for agricultural production. Although Arabidopsis (Arabidopsis thaliana) PHOSPHATE RESPONSE1 (PHR1) and its orthologs in other species have been shown to function in transcriptional regulation of phosphate (Pi) signaling and Pi homeostasis, an integrative comparison of PHR1-related proteins in rice (Oryza sativa) has not previously been reported. Here, we identified functional redundancy among three PHR1 orthologs in rice (OsPHR1, OsPHR2, and OsPHR3) using phylogenetic and mutation analysis. OsPHR3 in conjunction with OsPHR1 and OsPHR2 function in transcriptional activation of most Pi starvation-induced genes. Loss-of-function mutations in any one of these transcription factors (TFs) impaired root hair growth (primarily root hair elongation). However, these three TFs showed differences in DNA binding affinities and messenger RNA expression patterns in different tissues and growth stages, and transcriptomic analysis revealed differential effects on Pi starvation-induced gene expression of single mutants of the three TFs, indicating some degree of functional diversification. Overexpression of genes encoding any of these TFs resulted in partial constitutive activation of Pi starvation response and led to Pi accumulation in the shoot. Furthermore, unlike OsPHR2-overexpressing lines, which exhibited growth retardation under normal or Pi-deficient conditions, OsPHR3-overexpressing plants exhibited significant tolerance to low-Pi stress but normal growth rates under normal Pi conditions, suggesting that OsPHR3 would be useful for molecular breeding to improve Pi uptake/use efficiency under Pi-deficient conditions. We propose that OsPHR1, OsPHR2, and OsPHR3 form a network and play diverse roles in regulating Pi signaling and homeostasis in rice.

The Function of LPR1 is Controlled by an Element in the Promoter and is Independent of SUMO E3 Ligase SIZ1 in Response to Low Pi Stress in Arabidopsis thaliana

In Arabidopsis thaliana, there exist many typical responses to low phosphate (LP) stress, such as inhibition of primary root elongation, proliferation of lateral roots and accumulation of anthocyanin in leaves. The physiological, genetic and molecular mechanisms of these developmental responses remain undefined. We have isolated a phosphorus starvation-insensitive (psi) mutant. The mutant shows impaired inhibition of primary root growth, reduction of root hair growth and reduction of anthocyanin accumulation compared with the wild-type (WT) plants under an LP level. CycB1;1::GUS (cyclin B1;1::beta-glucuronidase) staining suggests that the mutant has a higher ability to maintain cell elongation and cell division than the WT. The genetic analysis and gene cloning indicate that psi is a new allele of lpr1 and that an AC-repeat element in the promoter plays important roles in controlling the expression of LPR1. The psi mutant also shows less sensitivity to auxin treatment compared with the WT and the mutant has an enhanced higher ability to maintain the auxin response in the root tip under LP. However, enhancing the auxin response in the quiescent center cannot mimic the mutant phenotype. These observations suggest that LPR1 is involved in the regulation of the auxin response to Pi starvation and auxin is probably not the only factor affected for maintaining the long-root phenotype under LP stress. Our results also indicate that the function of LPR1 is probably independent of SUMO E3 ligase SIZ1 in response to Pi starvation. The insensitive response of the psi mutant to brefeldin A suggests that LPR1 and PDR2 (Pi Deficiency Response 2) function in opposite ways in regulating the root growth response to Pi starvation in the endoplamic reticulum.

The Rice CK2 Kinase Regulates Trafficking of Phosphate Transporters in Response to Phosphate Levels

A kinase complex regulates phosphate transporter trafficking in response to phosphate, inhibiting interaction with the factor regulating exit from the endoplasmic reticulum to the plasma membrane. Phosphate transporters (PTs) mediate phosphorus uptake and are regulated at the transcriptional and posttranslational levels. In one key mechanism of posttranslational regulation, phosphorylation of PTs affects their trafficking from the endoplasmic reticulum (ER) to the plasma membrane. However, the kinase(s) mediating PT phosphorylation and the mechanism leading to ER retention of phosphorylated PTs remain unclear. In this study, we identified a rice (Oryza sativa) kinase subunit, CK2β3, which interacts with PT2 and PT8 in a yeast two-hybrid screen. Also, the CK2α3/β3 holoenzyme phosphorylates PT8 under phosphate-sufficient conditions. This phosphorylation inhibited the interaction of PT8 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1, a key cofactor regulating the exit of PTs from the ER to the plasma membrane. Additionally, phosphorus starvation promoted CK2β3 degradation, relieving the negative regulation of PT phosphorus-insufficient conditions. In accordance, transgenic expression of a nonphosphorylatable version of OsPT8 resulted in elevated levels of that protein at the plasma membrane and enhanced phosphorus accumulation and plant growth under various phosphorus regimes. Taken together, these results indicate that CK2α3/β3 negatively regulates PTs and phosphorus status regulates CK2α3/β3.

OsGLU3, a Putative Membrane-Bound Endo-1,4-Beta-Glucanase, Is Required for Root Cell Elongation and Division in Rice (Oryza sativa L.)

Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-β-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

Genetic manipulation of a high‑affinity PHR1 target cis‑element to improve phosphorous uptake in Oryza sativa L.

Phosphorus (P) is an essential macronutrient for crop development and production. Phosphate starvation response 1 (PHR1) acts as the central regulator for Pi-signaling and Pi-homeostasis in plants by binding to the cis-element PHR1 binding sequence (P1BS; GNATATNC). However, how phosphate starvation-induced gene expression is regulated remains obscure. In this work, we investigated the DNA binding affinity of the PHR1 ortholog OsPHR2 to its downstream target genes in Oryza sativa (rice). We confirmed that a combination of P1BS and P1BS-like motifs are essential for stable binding by OsPHR2. Furthermore, we report that variations in P1BS motif bases affected the binding affinity of OsPHR2 and that the highest affinity motif was GaATATtC (designated the A–T-type P1BS). We also found that a combination of two A–T-type P1BS elements in tandem, namely HA-P1BS, was very efficient for binding of OsPHR2. Using the cis-regulator HA-P1BS, we modified the promoters of Transporter Traffic Facilitator 1 (PHF1), a key factor controlling endoplasmic reticulum-exit of phosphate transporters to the plasma membrane, for efficient uptake of phosphorous in an energetically neutral way. Transgenic plants with the modified promoters showed significantly enhanced tolerance to low phosphate stress in both solution and soil conditions, which provides a new strategy for crop improvement to enhance tolerance of nutrient deficiency.