Dynamic interactions between SPX proteins, the ubiquitination machinery, and signalling molecules for stress adaptation at a whole-plant level.

The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF-CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.

The acyl-acyl carrier protein thioesterases GmFATA1 and GmFATA2 are essential for fatty acid accumulation and growth in soybean.

Acyl-acyl carrier protein (ACP) thioesterases (FAT) hydrolyze acyl-ACP complexes to release FA in plastids, which ultimately affects FA biosynthesis and profiles. Soybean GmFATA1 and GmFATA2 are homoeologous genes encoding oleoyl-ACP thioesterases whose role in seed oil accumulation and plant growth has not been defined. Using CRISPR/Cas9 gene editing mutation of Gmfata1 or 2 led to reduced leaf FA content and growth defect at the early seedling stage. In contrast, no homozygous double mutants were obtained. Combined this indicates that GmFATA1 and GmFATA2 display overlapping, but not complete functional redundancy. Combined transcriptomic and lipidomic analysis revealed a large number of genes involved in FA synthesis and FA chain elongation are expressed at reduced level in the Gmfata1 mutant, accompanied by a lower triacylglycerol abundance at the early seedling stage. Further analysis showed that the Gmfata1 or 2 mutants had increased composition of the beneficial FA, oleic acid. The growth defect of Gmfata1 could be at least partially attributed to reduced acetyl-CoA carboxylase activity, reduced abundance of five unsaturated monogalactosyldiacylglycerol lipids, and altered chloroplast morphology. On the other hand, overexpression of GmFATA in soybean led to significant increases in leaf FA content by 5.7%, vegetative growth, and seed yield by 26.9%, and seed FA content by 23.2%. Thus, overexpression of GmFATA is an effective strategy to enhance soybean oil content and yield.

Role of transcription factor complex OsbHLH156-OsIRO2 in regulating manganese, copper, and zinc transporters in rice.

Iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) are essential micronutrients that are necessary for plant growth and development, but can be toxic at supra-optimal levels. Plants have evolved a complex homeostasis network that includes uptake, transport, and storage of these metals. It was shown that the transcription factor (TF) complex OsbHLH156-OsIRO2 is activated under Fe deficient conditions and acts as a central regulator on Strategy II Fe acquisition. In this study, the role of the TF complex on Mn, Cu, and Zn uptake was evaluated. While Fe deficiency led to significant increases in shoot Mn, Cu, and Zn concentrations, the increases of these divalent metal concentrations were significantly suppressed in osbhlh156 and osiro2 mutants, suggesting that the TF complex plays roles on Mn, Cu, and Zn uptake and transport. An RNA-sequencing assay showed that the genes associated with Mn, Cu, and Zn uptake and transport were significantly suppressed in the osbhlh156 and osiro2 mutants. Transcriptional activation assays demonstrated that the TF complex could directly bind to the promoters of OsIRT1, OsYSL15, OsNRAMP6, OsHMA2, OsCOPT1/7, and OsZIP5/9/10, and activate their expression. In addition, the TF complex is required to activate the expression of nicotianamine (NA) and 2'-deoxymugineic acid (DMA) synthesis genes, which in turn facilitate the uptake and transport of Mn, Cu, and Zn. Furthermore, OsbHLH156 and OsIRO2 promote Cu accumulation to partially restore the Fe-deficiency symptoms. Taken together, OsbHLH156 and OsIRO2 TF function as core regulators not only in Fe homeostasis, but also in Mn, Cu, and Zn accumulation.

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.

Phosphate-dependent regulation of vacuolar trafficking of OsSPX-MFSs is critical for maintaining intracellular phosphate homeostasis in rice.

Vacuolar storage of inorganic phosphate (Pi) is essential for Pi homeostasis in plants. The SPX-MFS family proteins have been demonstrated to be vacuolar Pi transporters in many plant species. Transcriptional regulation of the predominant transporter among rice SPX-MFSs, OsSPX-MFS3, was only moderately suppressed by Pi starvation. Thus, post-transcriptional mechanisms were hypothesized to regulate the activity of OsSPX-MFS3. In this study, we found that the tonoplast localization of OsSPX-MFSs is inhibited under Pi-depleted conditions, resulting in their retention in the pre-vacuolar compartments (PVCs). A yeast two-hybrid screen identified that two SNARE proteins, OsSYP21 and OsSYP22, interact with the MFS domain of OsSPX-MFS3. Further genetic and cytological analyses indicate that OsSYP21 and OsSYP22 facilitate trafficking of OsSPX-MFS3 from PVCs to the tonoplast. Although a homozygous frameshift mutation in OsSYP22 appeared to be lethal, tonoplast localization of OsSPX-MFS3 was significantly inhibited in transgenic plants expressing a negative-dominant form of OsSYP22 (OsSYP22-ND), resulting in reduced vacuolar Pi concentrations in OsSYP22-ND plants. Under Pi-depleted conditions, the interaction between OsSYP22 and OsSPX-MFS3 was disrupted, and this process depended on the presence of the SPX domain. Deleting the SPX domains of OsSPX-MFSs resulted in their tonoplast localization under both Pi-depleted and Pi-replete conditions. Complementation of the osspx-mfs1/2/3 triple mutants with the MFS domain or the SPX domain of OsSPX-MFS3 confirmed that the MFS and SPX domains are responsive to Pi transport activity and Pi-dependent regulation, respectively. These data indicated that the SPX domains of OsSPX-MFSs sense cellular Pi (InsP) levels and, under Pi-depleted conditions, inhibit the interaction between OsSPX-MFSs and OsSYP21/22 and subsequent trafficking of OsSPX-MFSs from PVCs to the tonoplast.

High-resolution anatomical and spatial transcriptome analyses reveal two types of meristematic cell pools within the secondary vascular tissue of poplar stem.

The secondary vascular tissue emanating from meristems is central to understanding how vascular plants such as forest trees evolve, grow, and regulate secondary radial growth. However, the overall molecular characterization of meristem origins and developmental trajectories from primary to secondary vascular tissues in woody tree stems is technically challenging. In this study, we combined high-resolution anatomic analysis with a spatial transcriptome (ST) technique to define features of meristematic cells in a developmental gradient from primary to secondary vascular tissues in poplar stems. The tissue-specific gene expression of meristems and derived vascular tissue types were accordingly mapped to specific anatomical domains. Pseudotime analyses were used to track the origins and changes of meristems throughout the development from primary to secondary vascular tissues. Surprisingly, two types of meristematic-like cell pools within secondary vascular tissues were inferred based on high-resolution microscopy combined with ST, and the results were confirmed by in situ hybridization of, transgenic trees, and single-cell sequencing. The rectangle shape procambium-like (PCL) cells develop from procambium meristematic cells and are located within the phloem domain to produce phloem cells, whereas fusiform shape cambium zone (CZ) meristematic cells develop from fusiform metacambium meristematic cells and are located inside the CZ to produce xylem cells. The gene expression atlas and transcriptional networks spanning the primary transition to secondary vascular tissues generated in this work provide new resources for studying the regulation of meristem activities and the evolution of vascular plants. A web server (https://pgx.zju.edu.cn/stRNAPal/) was also established to facilitate the use of ST RNA-seq data.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean.

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

Co-regulation of mitochondrial and chloroplast function: Molecular components and mechanisms.

The metabolic interdependence, interactions, and coordination of functions between chloroplasts and mitochondria are established and intensively studied. However, less is known about the regulatory components that control these interactions and their responses to external stimuli. Here, we outline how chloroplastic and mitochondrial activities are coordinated via common components involved in signal transduction pathways, gene regulatory events, and post-transcriptional processes. The endoplasmic reticulum emerges as a point of convergence for both transcriptional and post-transcriptional pathways that coordinate chloroplast and mitochondrial functions. Although the identification of molecular components and mechanisms of chloroplast and mitochondrial signaling increasingly suggests common players, this raises the question of how these allow for distinct organelle-specific downstream pathways. Outstanding questions with respect to the regulation of post-transcriptional pathways and the cell and/or tissue specificity of organelle signaling are crucial for understanding how these pathways are integrated at a whole-plant level to optimize plant growth and its response to changing environmental conditions.

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.

Functional characterization of the three Oryza sativa SPX-MFS proteins in maintaining phosphate homoeostasis.

Plant vacuoles serve as the primary intracellular compartments for phosphorus (P) storage. The Oryza sativa genome contains three genes that encode SPX ( SYG1/ PHO81/ XPR1)-MFS ( Major Facility Superfamily) proteins (OsSPX-MFS1-3). The physiological roles of the three transporters under varying P conditions in laboratory and field are not known. To address this knowledge gap, we generated single, double and triple mutants for three OsSPX-MFS genes. All the mutants except Osspx-mfs2 display lower vacuolar Pi concentrations and OsSPX-MFSs overexpression plant display higher Pi accumulation, demonstrating that all OsSPX-MFSs are vacuolar Pi influx transporters. OsSPX-MFS3 plays the dominant role based on the phenotypes of single mutants in terms of growth, vacuolar and tissue Pi concentrations. OsSPX-MFS2 is the weakest and only functions as vacuole Pi sequestration in an Osspx-mfs1/3 background. The vacuolar Pi sequestration capacity was severely impaired in Osspx-mfs1/3 and Osspx-mfs1/2/3, which resulted in increased Pi allocation to aerial organs. High P in the panicle impaired panicle and fertility in Osspx-mfs1/3 and Osspx-mfs1/2/3. Osspx-mfs2 resulted in a more stable yield compared to the wild type under low P in field grown plants. The results suggest that alteration of vacuolar Pi sequestration may be a novel effective strategy to improve rice tolerance to low phosphorus in cropping systems.

PagDET2 promotes cambium cell division and xylem differentiation in poplar stem.

Secondary growth of the woody tree stem is governed by meristematic cell division and differentiation in the vascular cambium. Multiple hormonal signals and endogenous developmental programs regulate vascular cambium activity. Brassinosteroids (BRs) significantly promote secondary stem growth and wood formation in poplar trees. However, the underlying regulatory mechanisms of BRs within the vascular tissue remain unclear. Genetic and anatomical approaches were used here to elucidate the role of PagDET2, the rate-limiting enzyme for BRs biosynthesis, in regulating secondary vascular cambium activity in Populus. This study showed that the elevated endogenous castasterone (CS) levels in tree stems through overexpressing PagDET2 could enhance cambium meristem cell activity and xylem (XY) differentiation to promote secondary stem growth. RNA-seq analysis revealed that genes involved in BRs response, vascular cambium cell division, XY differentiation, and secondary cell wall synthesis were up-regulated.

DNA methylation is involved in acclimation to iron-deficiency in rice (Oryza sativa).

Iron (Fe) is an essential micronutrient in plants, and Fe limitation significantly affects plant growth, yield and food quality. While many studies have reported the transcriptomic profile and pursue molecular mechanism in response to Fe limitation, little is known if epigenetic factors play a role in response to Fe-deficiency. In this study, whole-genome bisulfite sequencing analysis, high-throughput RNA-Seq of mRNA, small RNA and transposable element (TE) expression with root and shoot organs of rice seedlings under Fe-sufficient and Fe-deficient conditions were performed. The results showed that widespread hypermethylation, especially for the CHH context, occurred after Fe-deficiency. Integrative analysis of methylation and transcriptome revealed that the transcript abundance of Fe-deficiency-induced genes was negatively correlated with nearby TEs and positively with the 24-nucleotide siRNAs. The ability of methylation to affect the physiology and molecular response to Fe-deficiency was tested using an exogenous DNA methyltransferase inhibitor (5-azacytidine), and genetically using a mutant for domains rearranged methyltransferase 2 (DRM2), that lacks CHH methylation. Both approaches resulted in decreased growth and Fe content in rice plants. Thus, alterations in specific methylation patterns, directed by siRNAs, play an important role in acclimation of rice to Fe-deficient conditions. Furthermore, comparison with other reports suggests this may be a universal mechanism to acclimate to limited nutrient availability.

CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds.

Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39-53% and 17-37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3-3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.

Transcriptomic Profiling of Fe-Responsive lncRNAs and Their Regulatory Mechanism in Rice.

Iron (Fe) deficiency directly affects crop growth and development, ultimately resulting in reduced crop yield and quality. Recently, long non-coding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in a multitude of pathways across numerous species. However, systematic screening of lncRNAs responding to Fe deficiency and their regulatory mechanism in plants has not been reported. In this work, 171 differently expressed lncRNAs (DE-lncRNAs) were identified based on analysis of strand-specific RNA-seq data from rice shoots and roots under Fe-deficient conditions. We also found several lncRNAs, which could generate miRNAs or act as endogenous target mimics to regulate expression of Fe-related genes. Analysis of interaction networks and gene ontology enrichment revealed that a number of DE-lncRNAs were associated with iron transport and photosynthesis, indicating a possible role of lncRNAs in regulation of Fe homeostasis. Moreover, we identified 76 potential lncRNA targets of OsbHLH156, a key regulator for transcriptional response to Fe deficiency. This study provides insight into the potential functions and regulatory mechanism of Fe-responsive lncRNAs and would be an initial and reference for any further studies regarding lncRNAs involved in Fe deficiency in plants.

Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing.

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/µL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

Analysis of Spatio-Temporal Transcriptome Profiles of Soybean (Glycine max) Tissues during Early Seed Development.

Soybean (Glycine max) is an important crop providing oil and protein for both human and animal consumption. Knowing which biological processes take place in specific tissues in a temporal manner will enable directed breeding or synthetic approaches to improve seed quantity and quality. We analyzed a genome-wide transcriptome dataset from embryo, endosperm, endothelium, epidermis, hilum, outer and inner integument and suspensor at the global, heart and cotyledon stages of soybean seed development. The tissue specificity of gene expression was greater than stage specificity, and only three genes were differentially expressed in all seed tissues. Tissues had both unique and shared enriched functional categories of tissue-specifically expressed genes associated with them. Strong spatio-temporal correlation in gene expression was identified using weighted gene co-expression network analysis, with the most co-expression occurring in one seed tissue. Transcription factors with distinct spatiotemporal gene expression programs in each seed tissue were identified as candidate regulators of expression within those tissues. Gene ontology (GO) enrichment of orthogroup clusters revealed the conserved functions and unique roles of orthogroups with similar and contrasting expression patterns in transcript abundance between soybean and Arabidopsis during embryo proper and endosperm development. Key regulators in each seed tissue and hub genes connecting those networks were characterized by constructing gene regulatory networks. Our findings provide an important resource for describing the structure and function of individual soybean seed compartments during early seed development.

OsIRO3 Plays an Essential Role in Iron Deficiency Responses and Regulates Iron Homeostasis in Rice.

Iron (Fe) homeostasis is essential for plant growth and development, and it is strictly regulated by a group of transcriptional factors. Iron-related transcription factor 3 (OsIRO3) was previously identified as a negative regulator for Fe deficiency response in rice. However, the molecular mechanisms by which OsIRO3 regulate Fe homeostasis is unclear. Here, we report that OsIRO3 is essential for responding to Fe deficiency and maintaining Fe homeostasis in rice. OsIRO3 is expressed in the roots, leaves, and base nodes, with a higher level in leaf blades at the vegetative growth stage. Knockout of OsIRO3 resulted in a hypersensitivity to Fe deficiency, with severe necrosis on young leaves and defective root development. The iro3 mutants accumulated higher levels of Fe in the shoot under Fe-deficient conditions, associated with upregulating the expression of OsNAS3, which lead to increased accumulation of nicotianamine (NA) in the roots. Further analysis indicated that OsIRO3 can directly bind to the E-box in the promoter of OsNAS3. Moreover, the expression of typical Fe-related genes was significantly up-regulated in iro3 mutants under Fe-sufficient conditions. Thus, we conclude that OsIRO3 plays a key role in responding to Fe deficiency and regulates NA levels by directly, negatively regulating the OsNAS3 expression.

Purple acid phosphatase 10c encodes a major acid phosphatase that regulates plant growth under phosphate-deficient conditions in rice.

Whilst constitutive overexpression of particular acid phosphatases (APases) can increase utilization of extracellular organic phosphate, negative effects are frequently observed in these transgenic plants under conditions of inorganic phosphate (Pi) sufficiency. In this study, we identified rice purple acid phosphatase 10c (OsPAP10c) as being a novel and major APase that exhibits activities associated both with the root surface and with secretion. Two constructs were used to generate the OsPAP10c-overexpression plants by driving its coding sequence with either a ubiquitin promoter (UP) or the OsPAP10c-native promoter (NP). Compared with the UP transgenic plants, lower expression levels and APase activities were observed in the NP plants. However, the UP and NP plants both showed a similar ability to degrade extracellular ATP and both promoted root growth. The growth performance and yield of the NP transgenic plants were better than the wild-type and UP plants in both hydroponic and field experiments irrespective of the level of Pi supply. Overexpression of APase by its native promoter therefore provides a potential way to improve crop production that might avoid increased APase activity in untargeted tissues and its inhibition of the growth of transgenic plants.

CASEIN KINASE2-Dependent Phosphorylation of PHOSPHATE2 Fine-tunes Phosphate Homeostasis in Rice.

Plants have evolved complex physiological and biochemical mechanisms to adapt to a heterogeneous soil phosphorus environment. PHOSPHATE2 (PHO2) is a phosphate (Pi) starvation-signaling regulator involved in maintaining Pi homeostasis in plants. Arabidopsis (Arabidopsis thaliana) PHO2 targets PHOSPHATE TRANSPORTER1 (PHT1) and PHO1 for degradation, whereas rice (Oryza sativa) PHO2 is thought to mediate PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 degradation. However, it is unclear whether and how PHO2 is post-translationally regulated. Here, we show that in rice, the CASEIN KINASE2 (OsCK2) catalytic subunit OsCK2α3 interacts with OsPHO2 in vitro and in vivo in vascular tissues cells, and phosphorylates OsPHO2 at Ser-841. Phosphorylated OsPHO2 is degraded more rapidly than native OsPHO2 in cell-free degradation assays. OsPHO2 interacts with OsPHO1 and targets it for degradation through a multivesicular body-mediated pathway. PHO1 mutation partially rescued the pho2 mutant phenotype. Further genetic analysis showed that a nonphosphorylatable version of OsPHO2 rescued the Ospho2 phenotype of high Pi accumulation in leaves better than native OsPHO2. In addition to the previously established role of OsCK2 in negatively regulating endoplasmic reticulum exit of PHT1 phosphate transporters, this work uncovers a role for OsCK2α3 in modulating Pi homeostasis through regulating the phosphorylation status and abundance of OsPHO2 in rice.