The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

Rice chromatin protein OsHMGB1 is involved in phosphate homeostasis and plant growth by affecting chromatin accessibility.

Although phosphorus is one of the most important essential elements for plant growth and development, the epigenetic regulation of inorganic phosphate (Pi) signaling is poorly understood. In this study, we investigated the biological function and mode of action of the high-mobility-group box 1 protein OsHMGB1 in rice (Oryza sativa), using molecular and genetic approaches. We determined that OsHMGB1 expression is induced by Pi starvation and encodes a nucleus-localized protein. Phenotypic analysis of Oshmgb1 mutant and OsHMGB1 overexpression transgenic plants showed that OsHMGB1 positively regulates Pi homeostasis and plant growth. Transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing indicated that OsHMGB1 regulates the expression of a series of phosphate starvation-responsive (PSR) genes by binding to their promoters. Furthermore, an assay for transposase-accessible chromatin followed by sequencing revealed that OsHMGB1 is involved in maintaining chromatin accessibility. Indeed, OsHMGB1 occupancy positively correlated with genome-wide chromatin accessibility and gene expression levels. Our results demonstrate that OsHMGB1 is a transcriptional facilitator that regulates the expression of a set of PSR genes to maintain Pi homeostasis in rice by increasing the chromatin accessibility, revealing a key epigenetic mechanism that fine-tune plant acclimation responses to Pi-limited environments.

Integrated transcriptomic analysis identifies coordinated responses to nitrogen and phosphate deficiency in rice.

Nitrogen (N) and phosphorus (P) are two primary components of fertilizers for crop production. Coordinated acquisition and utilization of N and P are crucial for plants to achieve nutrient balance and optimal growth in a changing rhizospheric nutrient environment. However, little is known about how N and P signaling pathways are integrated. We performed transcriptomic analyses and physiological experiments to explore gene expression profiles and physiological homeostasis in the response of rice (Oryza sativa) to N and P deficiency. We revealed that N and P shortage inhibit rice growth and uptake of other nutrients. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) suggested that N and Pi deficiency stimulate specific different physiological reactions and also some same physiological processes in rice. We established the transcriptional regulatory network between N and P signaling pathways based on all DEGs. We determined that the transcript levels of 763 core genes changed under both N or P starvation conditions. Among these core genes, we focused on the transcription factor gene NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) and show that its encoded protein is a positive regulator of P homeostasis and a negative regulator of N acquisition in rice. NIGT1 promoted Pi uptake but inhibited N absorption, induced the expression of Pi responsive genes PT2 and SPX1 and repressed the N responsive genes NLP1 and NRT2.1. These results provide new clues about the mechanisms underlying the interaction between plant N and P starvation responses.

Phenotypes and Molecular Mechanisms Underlying the Root Response to Phosphate Deprivation in Plants.

Phosphorus (P) is an essential macronutrient for plant growth. The roots are the main organ for nutrient and water absorption in plants, and they adapt to low-P soils by altering their architecture for enhancing absorption of inorganic phosphate (Pi). This review summarizes the physiological and molecular mechanisms underlying the developmental responses of roots to Pi starvation, including the primary root, lateral root, root hair, and root growth angle, in the dicot model plant Arabidopsis thaliana and the monocot model plant rice (Oryza sativa). The importance of different root traits and genes for breeding P-efficient roots in rice varieties for Pi-deficient soils are also discussed, which we hope will benefit the genetic improvement of Pi uptake, Pi-use efficiency, and crop yields.

A novel protein domain is important for photosystem II complex assembly and photoautotrophic growth in angiosperms.

Photosystem II (PSII) is a multi-subunit protein complex of the photosynthetic electron transport chain that is vital to photosynthesis. Although the structure, composition, and function of PSII have been extensively studied, its biogenesis mechanism remains less understood. Thylakoid rhodanese-like (TROL) provides an anchor for leaf-type ferredoxin:NADP+ oxidoreductase. Here, we report the chacterizaton of a second type of TROL protein, TROL2, encoded by seed plant genomes whose function has not previously been reported. We show that TROL2 is a PSII assembly cofactor with essential roles in the establishment of photoautotrophy. TROL2 contains a 45-amino-acid domain, termed the chlorotic lethal seedling (CLS) domain, that is both necessary and sufficient for TROL2 function in PSII assembly and photoautotrophic growth. Phylogenetic analyses suggest that TROL2 may have arisen from ancestral TROL1 via gene duplication before the emergence of seed plants and acquired the CLS domain via evolution of the sequence encoding its N-terminal portion. We further reveal that TROL2 (or CLS) forms an assembly cofactor complex with the intrinsic thylakoid membrane protein LOW PSII ACCUMULATION2 and interacts with small PSII subunits to facilitate PSII complex assembly. Collectively, our study not only shows that TROL2 (CLS) is essential for photoautotrophy in angiosperms but also reveals its mechanistic role in PSII complex assembly, shedding light on the molecular and evolutionary mechanisms of photosynthetic complex assemblyin angiosperms.

PROTEIN PHOSPHATASE95 Regulates Phosphate Homeostasis by Affecting Phosphate Transporter Trafficking in Rice.

Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (Oryza sativa), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing OsPP95 reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in ospp95 mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.

LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice.

Root system architecture is very important for plant growth and crop yield. It is essential for nutrient and water uptake, anchoring, and mechanical support. Root growth angle (RGA) is a vital constituent of root system architecture and is used as a parameter for variety evaluation in plant breeding. However, little is known about the underlying molecular mechanisms that determine root growth angle in rice (Oryza sativa). In this study, a rice mutant large root angle1 (lra1) was isolated and shown to exhibit a large RGA and reduced sensitivity to gravity. Genome resequencing and complementation assays identified OsPIN2 as the gene responsible for the mutant phenotypes. OsPIN2 was mainly expressed in roots and the base of shoots, and showed polar localization in the plasma membrane of root epidermal and cortex cells. OsPIN2 was shown to play an important role in mediating root gravitropic responses in rice and was essential for plants to produce normal RGAs. Taken together, our findings suggest that OsPIN2 plays an important role in root gravitropic responses and determining the root system architecture in rice by affecting polar auxin transport in the root tip.

OsHAC4 is critical for arsenate tolerance and regulates arsenic accumulation in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As(V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification.

OsHAC1;1 and OsHAC1;2 Function as Arsenate Reductases and Regulate Arsenic Accumulation.

Rice is a major dietary source of the toxic metalloid arsenic (As). Reducing its accumulation in rice (Oryza sativa) grain is of critical importance to food safety. Rice roots take up arsenate and arsenite depending on the prevailing soil conditions. The first step of arsenate detoxification is its reduction to arsenite, but the enzyme(s) catalyzing this reaction in rice remains unknown. Here, we identify OsHAC1;1 and OsHAC1;2 as arsenate reductases in rice. OsHAC1;1 and OsHAC1;2 are able to complement an Escherichia coli mutant lacking the endogenous arsenate reductase and to reduce arsenate to arsenite. OsHAC1:1 and OsHAC1;2 are predominantly expressed in roots, with OsHAC1;1 being abundant in the epidermis, root hairs, and pericycle cells while OsHAC1;2 is abundant in the epidermis, outer layers of cortex, and endodermis cells. Expression of the two genes was induced by arsenate exposure. Knocking out OsHAC1;1 or OsHAC1;2 decreased the reduction of arsenate to arsenite in roots, reducing arsenite efflux to the external medium. Loss of arsenite efflux was also associated with increased As accumulation in shoots. Greater effects were observed in a double mutant of the two genes. In contrast, overexpression of either OsHAC1;1 or OsHAC1;2 increased arsenite efflux, reduced As accumulation, and enhanced arsenate tolerance. When grown under aerobic soil conditions, overexpression of either OsHAC1;1 or OsHAC1;2 also decreased As accumulation in rice grain, whereas grain As increased in the knockout mutants. We conclude that OsHAC1;1 and OsHAC1;2 are arsenate reductases that play an important role in restricting As accumulation in rice shoots and grain.

LIGHT-INDUCED RICE1 Regulates Light-Dependent Attachment of LEAF-TYPE FERREDOXIN-NADP+ OXIDOREDUCTASE to the Thylakoid Membrane in Rice and Arabidopsis.

LIR1 (LIGHT-INDUCED RICE1) encodes a 13-kD, chloroplast-targeted protein containing two nearly identical motifs of unknown function. LIR1 is present in the genomes of vascular plants, mosses, liverworts, and algae, but not in cyanobacteria. Using coimmunoprecipitation assays, pull-down assays, and yeast two-hybrid analyses, we showed that LIR1 interacts with LEAF-TYPE FERREDOXIN-NADP(+) OXIDOREDUCTASE (LFNR), an essential chloroplast enzyme functioning in the last step of photosynthetic linear electron transfer. LIR1 and LFNR formed high molecular weight thylakoid protein complexes with the TIC62 and TROL proteins, previously shown to anchor LFNR to the membrane. We further showed that LIR1 increases the affinity of LFNRs for TIC62 and that the rapid light-triggered degradation of the LIR1 coincides with the release of the LFNR from the thylakoid membrane. Loss of LIR1 resulted in a marked decrease in the accumulation of LFNR-containing thylakoid protein complexes without a concomitant decrease in total LFNR content. In rice (Oryza sativa), photosynthetic capacity of lir1 plants was slightly impaired, whereas no such effect was observed in Arabidopsis thaliana knockout mutants. The consequences of LIR1 deficiency in different species are discussed.

OsCLT1, a CRT-like transporter 1, is required for glutathione homeostasis and arsenic tolerance in rice.

Arsenic (As) contamination in a paddy environment can cause phytotoxicity and elevated As accumulation in rice (Oryza sativa). The mechanism of As detoxification in rice is still poorly understood. We isolated an arsenate (As(V))-sensitive mutant of rice. Genomic resequencing and complementation identified OsCLT1, encoding a CRT-like transporter, as the causal gene for the mutant phenotype. OsCLT1 is localized to the envelope membrane of plastids. The glutathione and γ-glutamylcysteine contents in roots of Osclt1 and RNA interference lines were decreased markedly compared with the wild-type (WT). The concentrations of phytochelatin PC2 in Osclt1 roots were only 32% and 12% of that in WT after As(V) and As(III) treatments, respectively. OsCLT1 mutation resulted in lower As accumulation in roots but higher As accumulation in shoots when exposed to As(V). Under As(III) treatment, Osclt1 accumulated a lower As concentration in roots but similar As concentration in shoots to WT. Further analysis showed that the reduction of As(V) to As(III) was decreased in Osclt1. Osclt1 was also hypersensitive to cadmium (Cd). These results indicate that OsCLT1 plays an important role in glutathione homeostasis, probably by mediating the export of γ-glutamylcysteine and glutathione from plastids to the cytoplasm, which in turn affects As and Cd detoxification in rice.

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

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 rice CK2 kinase regulates trafficking of phosphate transporters in response to phosphate levels.

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, CK2ß3, which interacts with PT2 and PT8 in a yeast two-hybrid screen. Also, the CK2α3/ß3 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 CK2ß3 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/ß3 negatively regulates PTs and phosphorus status regulates CK2α3/ß3.

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.

Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner.

In plants, sensing the levels of external and internal nutrients is essential for reprogramming the transcriptome and adapting to the fluctuating environment. Phosphate (Pi) is a key plant nutrient, and a large proportion of Pi starvation-responsive genes are under the control of Phosphate Starvation Response Regulator 1 (PHR1) in Arabidopsis (AtPHR1) and its homologs, such as Oryza sativa (Os)PHR2 in rice. AtPHR1 and OsPHR2 expression is not very responsive to Pi starvation, raising the question as to how plants sense changes in cellular Pi levels to activate the central regulator. SPX [named after SYG1 (suppressor of yeast gpa1), Pho81 (CDK inhibitor in yeast PHO pathway), and XPR1 (xenotropic and polytropic retrovirus receptor)] proteins that harbor only the SPX domain are reported to be involved in the negative regulation of Pi starvation responses. Here, we show that the nuclear localized SPX proteins SPX1 and SPX2 are Pi-dependent inhibitors of the activity of OsPHR2 in rice. Indeed, SPX1 and SPX2 proteins interact with PHR2 through their SPX domain, inhibiting its binding to P1BS (the PHR1-binding sequence: GNATATNC). In vivo data, as well as results from in vitro experiments using purified SPX1, SPX2, and OsPHR2 proteins, showed that SPX1 and SPX2 inhibition of OsPHR2 activity is Pi-dependent. These data provide evidence to support the involvement of SPX1 and SPX2 in the Pi-sensing mechanism in plants.

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

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.

The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice.

The importance of SPX-domain-containing proteins to phosphate (Pi) homeostasis and signalling transduction has been established in plants. In this study, phylogenetic analysis revealed that OsSPX3 and OsSPX5 (SPX3/5) are paralogous SPX genes ( SYG1/Pho81/XPR1) in cereal crops. SPX3/5 are specifically responsive to Pi starvation at both the transcriptional and post-transcriptional levels. Similar tissue expression patterns of the two genes and proteins were identified by in situ hybridization and the transgenic plants harbouring SPX3pro-SPX3-GUS or SPX5pro-SPX5-GUS fusions, respectively. Both SPX3/5 are localized in the nucleus and cytoplasm in rice protoplasts and plants. SPX3/5 negatively regulate root-to-shoot Pi translocation with redundant function. The data showed that the Pi-starvation-accumulated SPX3/5 proteins are players in restoring phosphate balance following phosphate starvation. In vitro and in vivo protein-protein interaction analyses indicated that these two proteins can form homodimers and heterodimers, also implying their functional redundancy. Genetic interaction analysis indicated that SPX3/5 are functional repressors of OsPHR2 (PHR2), the rice orthologue of the central regulator AtPHR1 for Pi homeostasis and Pi signalling. These results suggest that the evolution of the additional redundant paralogous SPX genes is beneficial to plants recovering Pi homeostasis after Pi starvation by PHR2 pathway.

Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice.

We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High-affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two-electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and (33) P-labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild-type plants were observed under both high-Pi and low-Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double-knockdown plants grown under both high- and low-Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake.

Identification of a dual-targeted protein belonging to the mitochondrial carrier family that is required for early leaf development in rice.

A dual-targeted protein belonging to the mitochondrial carrier family was characterized in rice (Oryza sativa) and designated 3'-Phosphoadenosine 5'-Phosphosulfate Transporter1 (PAPST1). The papst1 mutant plants showed a defect in thylakoid development, resulting in leaf chlorosis at an early leaf developmental stage, while normal leaf development was restored 4 to 6 d after leaf emergence. OsPAPST1 is highly expressed in young leaves and roots, while the expression is reduced in mature leaves, in line with the recovery of chloroplast development seen in the older leaves of papst1 mutant plants. OsPAPST1 is located on the outer mitochondrial membrane and chloroplast envelope. Whole-genome transcriptomic analysis reveals reduced expression of genes encoding photosynthetic components (light reactions) in papst1 mutant plants. In addition, sulfur metabolism is also perturbed in papst1 plants, and it was seen that PAPST1 can act as a nucleotide transporter when expressed in Escherichia coli that can be inhibited significantly by 3'-phosphoadenosine 5'-phosphosulfate. Given these findings, together with the altered phenotype seen only when leaves are first exposed to light, it is proposed that PAPST1 may act as a 3'-phosphoadenosine 5'-phosphosulfate carrier that has been shown to act as a retrograde signal between chloroplasts and the nucleus.

OsORC3 is required for lateral root development in rice.

The origin recognition complex (ORC) is a pivotal element in DNA replication, heterochromatin assembly, checkpoint regulation and chromosome assembly. Although the functions of the ORC have been determined in yeast and model animals, they remain largely unknown in the plant kingdom. In this study, Oryza sativa Origin Recognition Complex subunit 3 (OsORC3) was cloned using map-based cloning procedures, and functionally characterized using a rice (Oryza sativa) orc3 mutant. The mutant showed a temperature-dependent defect in lateral root (LR) development. Map-based cloning showed that a G→A mutation in the 9th exon of OsORC3 was responsible for the mutant phenotype. OsORC3 was strongly expressed in regions of active cell proliferation, including the primary root tip, stem base, lateral root primordium, emerged lateral root primordium, lateral root tip, young shoot, anther and ovary. OsORC3 knockdown plants lacked lateral roots and had a dwarf phenotype. The root meristematic zone of ORC3 knockdown plants exhibited increased cell death and reduced vital activity compared to the wild-type. CYCB1;1::GUS activity and methylene blue staining showed that lateral root primordia initiated normally in the orc3 mutant, but stopped growing before formation of the stele and ground tissue. Our results indicate that OsORC3 plays a crucial role in the emergence of lateral root primordia.