The Amino Acid Transporter OsAAP4 Contributes to Rice Tillering and Grain Yield by Regulating Neutral Amino Acid Allocation through Two Splicing Variants

Abstract Background Amino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear. Results In this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (RNAi) and mutant lines displayed opposite trends compared with overexpresing (OE) lines. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering. Conclusion Our results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid allocation through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

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The amino acid transporter OsAAP4 contributes to rice tillering and grain yield by regulating neutral amino acid allocation through two splicing variants

10.21203/rs.3.rs-53363/v2 ◽

2020 ◽

Author(s):

Zhongming Fang ◽

Bowen Wu ◽

Yuanyuan Ji

Keyword(s):

Amino Acid ◽

Grain Yield ◽

Growth And Development ◽

Higher Plants ◽

Neutral Amino Acid ◽

Acid Uptake ◽

Amino Acid Permease ◽

Splicing Variant ◽

Rice Growth ◽

Splicing Variants

Abstract Background: Amino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear.Results: In this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (RNAi) and mutant lines displayed opposite trends compared with overexpresing (OE) lines. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering.Conclusion: Our results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid allocation through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

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The amino acid transporter OsAAP4 contributes to rice tillering and grain yield by regulating neutral amino acid allocation through two splicing variants

10.21203/rs.3.rs-53363/v3 ◽

2020 ◽

Author(s):

Zhongming Fang ◽

Bowen Wu ◽

Yuanyuan Ji

Keyword(s):

Amino Acid ◽

Grain Yield ◽

Growth And Development ◽

Higher Plants ◽

Neutral Amino Acid ◽

Acid Uptake ◽

Amino Acid Permease ◽

Splicing Variant ◽

Rice Growth ◽

Splicing Variants

Abstract Background: Amino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear. Results: In this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (RNAi) and mutant lines displayed opposite trends compared with overexpresing (OE) lines. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering. Conclusion: Our results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid allocation through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

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The amino acid transporter OsAAP4 contributes to rice tillering and grain yield by regulating neutral amino acid transport through two splicing variants

10.21203/rs.3.rs-53363/v1 ◽

2020 ◽

Author(s):

Zhongming Fang ◽

Bowen Wu ◽

Yuanyuan Ji

Keyword(s):

Amino Acid ◽

Grain Yield ◽

Amino Acid Transport ◽

Growth And Development ◽

Neutral Amino Acid ◽

Acid Uptake ◽

Acid Transport ◽

Splicing Variant ◽

Rice Growth ◽

Splicing Variants

Abstract Background Amino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear. Results In this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (Ri) and mutant lines displayed opposite trends. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering. Conclusion Our results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid transport through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

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The amino acid transporter AAP1 mediates growth and grain yield by regulating neutral amino acid uptake and reallocation in Oryza sativa

Journal of Experimental Botany ◽

10.1093/jxb/eraa256 ◽

2020 ◽

Vol 71 (16) ◽

pp. 4763-4777 ◽

Cited By ~ 2

Author(s):

Yuanyuan Ji ◽

Weiting Huang ◽

Bowen Wu ◽

Zhongming Fang ◽

Xuelu Wang

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Grain Yield ◽

Amino Acid Uptake ◽

Neutral Amino Acid ◽

Organic N ◽

Acid Uptake ◽

Amino Acid Permease ◽

Short Palindromic Repeat ◽

Exogenous Treatment

Abstract Nitrogen (N) is a major element necessary for crop yield. In most plants, organic N is primarily transported in the form of amino acids. Here, we show that amino acid permease 1 (AAP1) functions as a positive regulator of growth and grain yield in rice. We found that the OsAAP1 gene is highly expressed in rice axillary buds, leaves, and young panicles, and that the OsAAP1 protein is localized to both the plasma membrane and the nuclear membrane. Compared with the wild-type ZH11, OsAAP1 overexpression (OE) lines exhibited increased filled grain numbers as a result of enhanced tillering, while RNAi and CRISPR (clustered regularly interspaced short palindromic repeat; Osaap1) knockout lines showed the opposite phenotype. In addition, OsAAP1-OE lines had higher concentrations of neutral and acidic amino acids, but lower concentrations of basic amino acids in the straw. An exogenous treatment with neutral amino acids promoted axillary bud outgrowth more strongly in the OE lines than in the WT, RNAi, or Osaap1 lines. Transcriptome analysis of Osaap1 further demonstrated that OsAAP1 may affect N transport and metabolism, and auxin, cytokinin, and strigolactone signaling in regulating rice tillering. Taken together, these results support that increasing neutral amino acid uptake and reallocation via OsAAP1 could improve growth and grain yield in rice.

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Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218794627 ◽

2018 ◽

Vol 24 (2) ◽

pp. 111-120 ◽

Cited By ~ 6

Author(s):

Sanjay J. Danthi ◽

Beirong Liang ◽

Oanh Smicker ◽

Benjamin Coupland ◽

Jill Gregory ◽

...

Keyword(s):

Amino Acid ◽

High Throughput Screening ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Imaging Plate ◽

Acid Uptake ◽

Functional Assays ◽

Neutral Amino Acid Transporter ◽

Acid Transporter ◽

Pharmacologically Active

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

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Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

Journal of Bacteriology ◽

10.1128/jb.184.15.4071-4080.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4071-4080 ◽

Cited By ~ 76

Author(s):

A. H. F. Hosie ◽

D. Allaway ◽

C. S. Galloway ◽

H. A. Dunsby ◽

P. S. Poole

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Rhizobium Leguminosarum ◽

Binding Protein ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Amino Acid Permease ◽

Branched Chain Amino Acid ◽

General Amino Acid Permease ◽

Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

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