OsNPF3.1,a nitrate,abscisic acid and gibberellin transporter gene,is essential for rice tillering and nitrogen utilization efficiency

Low-affinity nitrate transporter genes have been identified in subfamilies 4-8 of the rice nitrate transporter 1(NRT1)/peptide transporter family(NPF),but the OsNPF3 subfamily responsible for nitrate and phytohormone transport and rice growth and development remains unknown.In this study,we described OsNPF3.1 as an essential nitrate and phytohormone transporter gene for rice tillering and nitrogen utilization efficiency(NUtE).OsNPF3.1 possesses four major haplotypes of its promoter sequence in 517 cultivars,and its expression is positively associated with tiller number.Its expression was higher in the basal part,culm,and leaf blade than in other parts of the plant,and was strongly induced by nitrate,abscisic acid(ABA)and gibberellin 3(GA_3)in the root and shoot of rice.Electrophysiological experiments demonstrated that OsNPF3.1 is a pH-dependent low-affinity nitrate transporter,with rice protoplast uptake assays showing it to be an ABA and GA_3 transporter.OsNPF3.1 overexpression significantly promoted ABA accumulation in the roots and GA accumulation in the basal part of the plant which inhibited axillary bud outgrowth and rice tillering,especially at high nitrate concentrations.The NUtE of OsNPF3.1-overexpressing plants was enhanced under low and medium nitrate concentrations,whereas the NUtE of OsNPF3.1 clustered regularly interspaced short palindromic repeats(CRISPR)plants was increased under high nitrate concentrations.The results indicate that OsNPF3.1 transports nitrate and phytohormones in different rice tissues under different nitrate concentrations.The altered OsNPF3.1 expression improves NUtE in the OsNPF3.1-overexpressing and CRISPR lines at low and high nitrate concentrations,respectively.

Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Peptide Transporter OsNPF8.1 Contributes to Sustainable Growth under Salt and Drought Stresses,and Grain Yield under Nitrogen Deficiency in Rice

Peptide transport is important for plant tissues where rapid proteolysis occurs,especially during germination and senescence,to enhance redistribution of organic nitrogen(N).However,the biological role of peptide transporters is poorly investigated in rice.We characterized the function of the peptide transporter OsNPF8.1 of rice nitrate transporter 1/peptide transporter family(NPF).Ectopic expression of OsNPF8.1 in yeast revealed that OsNPF8.1 encoded a high-affinity di-/tri-peptide transporter,and the osnpf8.1 mutants had a lower uptake rate of the fluorescent-labelled dipeptide c in leaves of rice seedlings.Histochemical assays showed that OsNPF8.1 was highly expressed in mesophyll cells and vascular parenchyma cells,but not detected in root hairs and epidermises.Expression of OsNPF8.1 was induced by N deficiency,drought,Na Cl and abscisic acid,and kept at a high level in senescing leaves.Under N deficiency conditions,compared with the wild type Zhonghua 11,the osnpf8.1 mutants grew slower at the seedling stage,and had lower grain yield and lower N content in the grains.In contrast,OsNPF8.1-over-expressing rice(OsNPF8.1-OE)grew faster at the seedling stage and had a higher grain yield.The osnpf8.1 seedlings were less tolerant to salt and drought stresses.These results suggested that stress-induced organic N transportation mediated by OsNPF8.1 might contribute to balance plant growth and tolerate to salt/drought stress and N-deficiency.

Molecular Basis and Regulation of Ammonium Transporter in Rice

Rice grows in flooded paddy fields and takes up ammonium as the preferred nitrogen （N） source. Ammonium uptake is facilitated by a family of integral membrane proteins known as ammonium transporters found in all domains of life. However, the molecular mechanism and functional characteristics of the ammonium transporters （AMT） in rice have not been determined in detail yet. In this review, we report a genome-wide search for AMT genes in rice, resulting in the increase of the number of potential AMT proteins to at least 12, including members of both the alpha and beta sub-groups. Analysis of the predicted protein sequences for the 12 OsAMT proteins identified many conserved phosphorylation sites in both the alpha and beta group members, which could potentially play a role in controlling the activity of the transporters. Present knowledge of the expression of rice AMT genes is also summarized in detail. Future studies should focus on the structural and functional characteristics of OsAMT proteins to provide insight into the mechanism of ammonium uptake and its regulation in rice. Such research could improve utilization and decrease wastage of N fertilizer in rice cultivation.

Molecular cloning and expression analysis of turnip (Brassica rapa var. rapa) sucrose transporter gene family

In higher plants, sugars(mainly sucrose) are produced by photosynthetically assimilated carbon in mesophyll cells of leaves and translocated to heterotrophic organs to ensure plant growth and development. Sucrose transporters, or sucrose carriers(SUCs), play an important role in the long-distance transportation of sucrose from source organs to sink organs, thereby affecting crop yield and quality.The identification, characterization, and molecular function analysis of sucrose transporter genes have been reported for monocot and dicot plants. However, no relevant study has been reported on sucrose transporter genes in Brassica rapa var. rapa, a cruciferous root crop used mainly as vegetables and fodder.We identified and cloned 12 sucrose transporter genes from turnips, named BrrSUC1.1 to BrrSUC6.2according to the SUC gene sequences of B. rapa pekinensis. We constructed a phylogenetic tree and analyzed conserved motifs for all 12 sucrose transporter genes identified. Real-time quantitative polymerase chain reaction was conducted to understand the expression levels of SUC genes in different tissues and developmental phases of the turnip. These findings add to our understanding of the genetics and physiology of sugar transport during taproot formation in turnips.

OsNPF5.16, a nitrate transporter gene with natural variation, is essential for rice growth and yield

Rice has a large number of nitrate or peptide transporter family(NPF) genes, but the effects of most members on rice growth and development are unknown. We report that Os NPF5.16, a nitrate transporter gene with natural variation in its promoter sequence, is essential for rice growth and yield. The promoter sequence showed various differences between indica and japonica cultivars, and higher expression of Os NPF5.16 was found in indica cultivars with higher plant weight and more tillers than japonica cultivars.Os NPF5.16 was highly expressed in roots, tiller basal parts, and leaf sheaths, and its protein was localized on the plasma membrane. In c RNA-injected Xenopus laevis oocytes, Os NPF5.16 transport of nitrate at high nitrate concentration depended on p H. Overexpression of Os NPF5.16 increased nitrate content and total nitrogen content in leaf sheath as well as biomass and tiller bud length in rice. Elevated expression of Os NPF5.16 increased rice tiller number and grain yield by regulating cytokinin levels. Inhibition of Os NPF5.16 expression showed the opposite effects. Regulating Os NPF5.16 expression has potential for improving rice grain yield.

Less and shrunken pollen 1(LSP1) encodes a member of the ABC transporter family required for pollen wall development in rice(Oryza sativa L.)

Pollen fertility is an agronomic trait that strongly influences rice yield. Recent studies have revealed that the development of the pollen wall is required for pollen fertility and is regulated by several genes. However, the mechanisms underlying pollen and pollen wall development in rice remain largely unknown. In the present study, a point mutation in a gene on chromosome 1 was identified that resulted in the production of less and shrunken pollen(LSP) and led to defects in pollen wall formation. This gene was named LSP1 and was found to encode a member of the adenosine triphosphate-binding cassette(ABC)transporter G subfamily, OsABCG3. Two other loss-of-function mutants of LSP1/OsABCG3,generated using CRISPR/Cas9 technology, showed the same male sterile phenotype. The LSP1/OsABCG3 gene showed a spatio-temporal expression pattern in the developing anthers, and is an ortholog of the Arabidopsis genes At ABCG1 and At ABCG16, which play an important role in pollen wall development. Mutation of LSP1/OsABCG3 affected the expression of several genes involved in pollen and pollen wall formation. These results suggest that LSP1/OsABCG3 is critical for normal pollen fertility and shed light on the molecular mechanisms underlying rice pollen wall development.

Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.

A mitochondrial phosphate transporter,McPht gene,confers an acclimation regulation of the transgenic rice to phosphorus deficiency

Phosphate transporters play an important role in promoting the uptake and transport of phosphate in plants. In this study, the McPht gene from the Mesembryanthemum crystallinum, a mitochondrial phosphate transporter, was isolated and constructed onto a constitutive expression vector carrying 35S：：GFP, and the recombinant constructs were transferred into Oryza sativajaponica L. cv. Kitaake to investigate the regulatory role of the McPhtgene under phosphorus deficiency. The McPhtgene encodes a protein of 357 amino acids with six transmembrane domains and is located to the mitochondria, and the mRNAtranscripts of the McPht gene are highly accumulated in the shoots of M. crystallinum in response to phosphorus deficiency. However, more mRNA transcripts of the McPht gene were accumulated in the roots of the transgenic rice under phosphorus deficiency. Measurements showed that the transgenic rice demonstrated an enhanced promotion in the root development, the root activities, and phosphate uptake under phosphorus deficiency. Transcriptome sequencing showed that the transgenic rice exhibited total of 198 differentially expressed genes. Of these, total of 154 differentially expressed genes were up-regulated and total 44 genes were down-regulated comparing to the wild type in response to phosphorus deficiency. The selective six genes of the up-regulated differentially expressed genes showed an enhanced increase in mRNA transcripts in response to phosphorus deficiency, however, the transcripts of the mitochondrial carrier protein transporter in rice, a homologous gene of the McPht, in both the transgenic line and the wild type had no obvious differences. Functional enrichment analyses revealed that the most of the up-regulated genes are involved in the cytoplasmic membrane-bounded vesicle, and most of the down-regulated genes are involved in the mitochondrion and cytoplasmic membrane-bounded vesicle. The differentially expressed genes were highly enriched in plant secondary metabolisms and plant-pathogen interaction. These results indicated that the overexpression of the McPht gene might participate in the physiological adaptive modulation of the transgenic rice to phosphorus deficiency by up- or down-regulating the differentially expressed genes.

OsbZIP72 Is Involved in Transcriptional Gene-Regulation Pathway of Abscisic Acid Signal Transduction by Activating Rice High-Affinity Potassium Transporter OsHKT1;1

We created CRISPR-Cas9 knock-out and overexpressing OsbZIP72 transgenic rice plants to gain a better understanding of the role and molecular mechanism of OsbZIP72 gene in stress tolerance,which has remained largely elusive.OsbZIP72 was expressed and integrated into rice transgenic plant genomes,and the OsbZIP72 transcript in overexpression lines was elicited by salinity,abscisic acid(ABA)and drought stresses.OsbZIP72 overexpressing plants showed higher tolerance to drought and salinity stresses,while knock-out transgenic lines showed higher sensitivity to these stresses.The differentially expressed genes(DEGs)from RNA-sequencing data encompassed several abiotic stress genes,and the functional classification of these DEGs demonstrated the robust transcriptome diversity in OsbZIP72.Yeast one-hybrid,along with luciferase assay,indicated that OsbZIP72 acted as a transcriptional initiator.Remarkably,electrophoresis mobility assay revealed that OsbZIP72 bound directly to the ABAresponsive element in the OsHKT1;1 promoter region and activated its transcription.Overall,our findings revealed that OsbZIP72 can act as a transcriptional modulator with the ability to induce the expression of OsHKT1;1 in response to environmental stress through an ABA-dependent regulatory pathway,indicating that OsbZIP72 can play a crucial role in the ABA-mediated salt and drought tolerance pathway in rice.

Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains under Two Establishment Methods

Six rice varieties, PR120, PR116, Feng Ai Zan, PR115, PAU201 and Punjab Mehak 1 were raised under aerobic and transplanting conditions to assess the effects of planting conditions on sucrose metabolising enzymes in relation to the transformation of free sugars to starch and protein in flag leaves and grains. Activities of sucrose synthase, sucrose phosphate synthase and acid invertase increased till flowering stage in leaves and mid-milky stage（14 d after flowering） in grains and thereafter declined in concomitant with the contents of reducing sugar. Under aerobic conditions, the activities of acid invertase and sucrose synthase（cleavage） significantly decreased in conjunction with the decrease in non-reducing sugars and starch content in all the varieties. Disruption of starch biosynthesis under the influence of aerobic conditions in both leaves and grains and the higher build up of sugars possibly resulted in their favoured utilization in nitrogen metabolism. Feng Ai Zan, PR115 and PR120 maintained higher levels of sucrose synthase enzymes in grains and leaves and contents of metabolites（amino acid, protein and non-reducing sugar） under aerobic conditions, while PR116, Punjab Mehak 1 and PAU201 performed better under transplanting conditions, thus showing their adaptation to environmental stress. Yield gap between aerobic and transplanting rice is attributed primarily to the difference in sink activity and strength. Overall, it appear that up-regulation of sucrose synthase（synthesis） and sucrose phosphate synthase under aerobic conditions might be responsible in enhancing growth and productivity of rice varieties.

Functional Analysis of Phosphate Transporter OsPHT4 Family Members in Rice

The transport of phosphate between cytoplasm and subcellular compartments is critical for plant metabolic regulation.We conducted bioinformatic analysis,heterologous expression in yeast,gene expression pattern and subcellular localization analysis to characterize the possible functions of OsPHT4 gene family in rice.Together with the PHT4 genes from higher plants,OsPHT4s can be classified into six distinct groups.OsPHT4;1-OsPHT4;4 are targeted to chloroplasts,and OsPHT4;6-1 and OsPHT4;6-2 are located to Golgi apparatus.OsPHT4 proteins can mediate inorganic phosphate(Pi)transport in yeast.In addition,dynamic transcriptional changes determined by qRT-PCR revealed different expression profiles of OsPHT4 genes in response to phosphate starvation,salicylic acid,abscisic acid and salt stress treatments.These results suggested that OsPHT4 proteins are involved in Pi distribution between the cytoplasm and chloroplast or Golgi apparatus and also involved in stress responses.

Characterization and functional divergence of genes encoding sucrose transporters in oilseeds castor bean

Castor bean(Ricinus communis L.)is an economically important non-edible oilseed crop.Its seed oils are rich in hydroxy fatty acid,which are highly valuable with a wide range of industrial applications.Sucrose transportation is critical in regulating the growth,development and oilseed yield in castor bean.The transporters or carriers(SUTs or SUCs)play a central role in orchestrating sucrose allocation and aiding in plant adaptation to diverse stresses.In this study,based on castor bean genome,three RcSUCs(RcSUC2,RcSUC3 and RcSUC4)were identified and characterized.The expressional profiles of RcSUCs in different tissues such as leaf,stem,root,phloem and seed tissues exhibited a distinct divergence of gene expression,suggesting that the functions of RcSUC2,RcSUC3 and RcSUC4 are differentiated into long or short-distance transportation among tissues.Additionally,under abiotic stresses including hot temperature,low temperature,drought and salt stresses,the sugar allocation among leaf,stem and roots was tested.The expressional changes of Rc SUCs in leaf,stem and root tissues were associated with sugar transportation and allocation.Taken together,the differential expression of Rc SUCs among tissues responsing to abiotic stress suggested functional differences in sucrose transport and redistribution in different tissues.This study is helpful to understand the physiological and molecular mechanisms of sucrose transportation and allocation among tissues in heterotrophic oilseeds,and could provide clues for genetic improvement and optimization of cultivation practices.

Polyphosphate Accelerates Transformation of Nonstructural Carbohydrates to Improve Growth of ppk-Expressing Transgenic Rice in Phosphorus Deficiency Culture

Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils,a key limiting factor for sustainable development in modern agriculture.The polyphosphate kinase(ppk)gene-expressing transgenic rice with a single-copy line(ETRS)is constructed to improve phosphate fertilizer utilization efficiency for phosphorus resource conservation.To investigate the potential mechanisms of the increased biomass in ETRS in low phosphate culture,ETRS was cultivated in a low inorganic phosphate(Pi)culture medium(15μmol/L Pi,LP)and a normal Pi culture medium(300μmol/L Pi,CP),respectively.After 89 d of cultivation in different concentrations of phosphate culture media,the total phosphorus,polyphosphate(polyP),biomass,photosynthetic rate,nonstructural carbohydrate(NSC)contents,related enzyme activities,and related gene expression levels were analyzed.The results showed that ETRS had a high polyP amount to promote the photosynthetic rate in LP,and its biomass was almost the same as the wild type(WT)in CP.The NSC content of ETRS in LP was higher than that of WT in LP,but slightly lower than that of WT in CP.PolyP notably promoted the sucrose phosphate synthase activities of ETRS and significantly down-regulated the expression levels of sucrose transporter genes(OsSUT3 and OsSUT4),resulting in inhibiting the transport of sucrose from shoot to root in ETRS.It was concluded that polyP can stimulate the synthesis of NSCs in LP,which improved the growth of ETRS and triggered the biological activities of ETRS to save phosphate fertilizer.Our study provides a new way to improve the utilization rate of phosphate fertilizer in rice production.

Expression Pattern of Sucrose Transporters in <i>Arabidopsis thaliana</i>During Aphid (<i>Myzus persicae</i>) Infestation

Herbivorous insects change the metabolism of the plant during their attack. Our study reports the changes in the expression pattern of sucrose transporters in response to the infestation of aphids at different time intervals. Results showed a significant enhancement in the expression pattern for six out of nine sucrose transporters in response to aphid infestation, followed by suppression after some point. During an earlier time point of infestation, the expressions of sucrose transporters were enhanced probably to compensate for the energy requirements of the damaged cell. However, suppression of sucrose transporters at a later stage may be a defense strategy of the plant to repel the aphids because at a later stage of infestation, aphids become a secondary sink. To complement our assumption, we performed aphid infestation choice and reproductive performance tests in the null mutant of one of the transporters, SUC2, which was compromised in phloem loading of sucrose. Results showed that the mutant was less preferable to aphid for choice as well as reproduction performance.

Isolation and cloning of the gene encoding high affinity phosphate transporter in rice

High affinity phosphate transporterplays an important role in plantadapting to low phosphorus. Isolationof genes coding this kind of proteinhas attracted worldwide scholars toaccomplish. We aimed to isolate thegene and transfer it to target plants

<i>ABNORMAL SHOOT IN YOUTH</i>, a Homolog of Molybdate Transporter Gene, Regulates Early Shoot Development in Rice

We analyzed the abnormal shoot in youth (asy) mutant to understand the phase-specific regulation of shoot development. asy showed various shoot abnormalities, including small leaves due to the precocious termination of cell division, defects in leaf blade-sheath boundary formation, and abnormal shoot apical meristem maintenance at the early vegetative stage. These defects recovered with advanced development. ASY encodes a DUF791 domain protein, which is part of the major facilitator superfamily. Despite stage-specific phenotypes, the ASY expression level was roughly constant throughout development. A paralog of ASY, ASL, exists in the rice genome and is supposed to have redundant functions. ASL expression was relatively low in early-stage embryos but increased at later stages. Thus, asy phenotypes were limited to the stage when ASL expression was suppressed. A homology search revealed that ASY is a homolog of the Chlamydomonas CrMoT2 gene, which encodes a molybdate transporter. ASY was suggested to encode a molybdate transporter based on its sequence similarity with CrMoT2 and predicted transmembrane topology. This is the first report of a CrMOT2-type molybdate transporter in higher plants.

Knocking-Out OsPDR7 Triggers Up-Regulation of OsZIP9 Expression and Enhances Zinc Accumulation in Rice

Zinc(Zn) is an essential trace mineral that is required for plant growth and development. A number of protein transporters, which are involved in Zn uptake, translocation and distribution, are finely regulated to maintain Zn homeostasis in plant. In this study, we functionally characterized an ATP-binding cassette(ABC) transporter gene, OsPDR7, which is involved in Zn homeostasis. Os PDR7 encodes a plasma membrane-localized protein that is expressed mainly in the exodermis and xylem in the rice root.ospdr7 mutants resulted in higher Zn accumulation compared with the wild type. Heterogeneous expression of OsPDR7 in a yeast mutant rescued the Zn-deficiency phenotype, implying transport activity of OsPDR7 to Zn in yeast. However, no ZIP genes except for OsZIP9 showed change in expression profile in the ospdr7 mutants, which suggested that OsPDR7 maintains cellular Zn homeostasis through regulating Os ZIP9 expression. RNA-Seq analysis further revealed a set of differentially expressed genes between the wild type and ospdr7 mutants that allowed us to propose a possible OsPDR7-associated signaling network involving transporters, hormone responsive genes, and transcription factors. Our results revealed a novel transporter involved in the regulation of Zn homeostasis and will pave the way toward a better understanding of the fine-tuning of gene expression in the network of transporter genes.

A Pleiotropic Drug Resistance Family Protein Gene Is Required for Rice Growth, Seed Development and Zinc Homeostasis

Zinc(Zn) is an essential mineral element for plant growth and development. Zn deficiency in crops frequently occurs in many types of soils. It is therefore crucial to identify genetic resources linking Zn acquisition traits and development of crops with improved Zn-use efficiency for sustainable crop production. In this study, we functionally identified a rice uncharacterized ABCG(ATP-binding cassette G-subfamily) gene encoding a PDR20(pleiotropic drug resistance 20) metal transporter for mediation of rice growth, seed development and Zn accumulation. OsPDR20 was localized to the plasma membrane, but it was not transcriptionally induced under Zn deficiency, rather was sufficiently up-regulated under high level of Zn stress. Yeast(Saccharomyces cerevisiae) transformed with OsPDR20 displayed a relatively lower Zn accumulation with attenuated cellular growth, suggesting that OsPDR20 had an activity for Zn transport. Knocking-down OsPDR20 by RNA interference(RNAi) compromised rice growth with shorter plant height and decreased biomass in rice plantlets grown under hydroponic media. Zn concentration in the roots of OsPDR20 knocked-down rice lines declined under Zn deficiency, while they remained unchanged compared with the wild type under normal Zn supply. A rice lifelong field trial demonstrated that OsPDR20 mutation impaired the capacity of seed development, with shortened panicle and seed length, compromised spikelet fertility, and reduced grain number per plant or grain weight per unit area. Interestingly, OsPDR20 mutation elevated the accumulation of Zn in husk and brown rice over the wild type. Overall, this study pointed out that OsPDR20 is fundamentally required for rice growth and seed development through Zn transport and homeostasis.