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OsAAP1 positively regulates growth and grain yield in rice by influencing amino acid uptake and reallocation., 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.

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.

Additional file 2: Figure S2. Sequencing of two different types of promoter sequences of OsAAP4. NIP indicates Nipponbare, C172 indicates the Hap5 type in Japonica. W144 indicates the Hap2 type in Indica.

Additional file 14 Table S1. List of the primers in this study.

Summary Amino acid transporters (AATs) play indispensable roles in nutrient allocation during plant development. In this study, we demonstrated that inhibiting expression of the rice amino acid transporter OsAAP3 increased grain yield due to a formation of larger numbers of tillers as a result of increased bud outgrowth. Elevated expression of OsAAP3 in transgenic plants resulted in significantly higher amino acid concentrations of Lys, Arg, His, Asp, Ala, Gln, Gly, Thr and Tyr, and inhibited bud outgrowth and rice tillering. However, RNAi of OsAAP3 decreased significantly Arg, Lys, Asp and Thr concentrations to a small extent, and thus promoted bud outgrowth, increased significantly tiller numbers and effective panicle numbers per plant, and further enhanced significantly grain yield and nitrogen use efficiency (NUE). The promoter sequences of OsAAP3 showed some divergence between Japonica and Indica rice, and expression of the gene was higher in Japonica, which produced fewer tillers than Indica. We generated knockout lines of OsAAP3 on Japonica ZH11 and KY131 using CRISPR technology and found that grain yield could be increased significantly. These results suggest that manipulation of OsAAP3 expression could be used to increase grain yield in rice.

Rice tillering, a key architecture trait determining grain yield, is highly regulated by a class of newly identified phytohormones, strigolactones (SLs). However, the whole SL signaling pathway from the receptor to downstream transcription factors to finally inhibit tillering remains unrevealed. In this study, we first found that brassinosteroids (BRs) strongly enhance tillering by promoting bud outgrowth in rice, which is largely different from the function of BRs in Arabidopsis. Genetic and biochemical analyses indicated that both the SL and BR signaling pathways control rice tillering by regulating the stability of D53 and/or the OsBZR1–RLA1–DLT module, a transcriptional complex in the rice BR signaling pathway. We further found that D53 interacts with OsBZR1 to inhibit the expression of FC1, a local inhibitor of tillering, and that this inhibition depends on direct DNA binding by OsBZR1, which recruits D53 to the FC1 promoter in rice buds. Taken together, these findings uncover a mechanism illustrating how SLs and BRs coordinately regulate rice tillering via the early responsive gene FC1.

As fundamental nutrients, amino acids are important for rice (Oryza sativa) growth and development. Here, we identified the amino acid permease 5 (OsAAP5), that regulates tiller number and grain yield in rice. The OsAAP5 promoter sequence differed between indica and japonica rice varieties. Lower expression of OsAAP5 in the young leaf blade in indica varieties than in japonica varieties was associated with more tillers in indica than in japonica. Down-regulation of OsAAP5 expression in japonica using RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats led to increases in tiller number and grain yield, whereas OsAAP5 overexpression (OE) had the opposite effect. Both a protoplast amino acid uptake assay and HPLC analysis indicated that more basic (Lys, Arg) and neutral (Val, Ala) amino acids were transported and accumulated in the OE lines than in the wild type, but the opposite was observed in the RNAi lines. Furthermore, exogenous application of Lys, Arg, Val, and Ala in the OE lines substantially inhibited tiller bud elongation, but the effect was lost in the RNAi lines. Notably, concentrations of the cytokinins cis-zeatin and dihydrozeatin were much lower in the OE lines than in the wild type, whereas concentrations in the RNAi lines were higher. Thus, OsAAP5 could regulate tiller bud outgrowth by affecting cytokinin levels, and knockout of OsAAP5 could be valuable for japonica breeding programs seeking high yield and grain quality.

Background Rice tiller number is one of the most important factors that determine grain yield, while nitrogen is essential for the crop growth and development, especially for tiller formation. Genes involved in nitrogen use efficiency processes have been identified in the previous studies, however, only a small number of these genes have been found to improve grain yield by promoting tillering. Results We constructed over-expression (OX) lines and RNA-interference (Ri) lines, and selected a mutant of OsNPF7.2, a low-affinity nitrate transporter. Our analyses showed that rice tiller number and grain yield were significantly increased in OX lines, whereas Ri lines and mutant osnpf7.2 had fewer tiller number and lower grain yield. Under different nitrate concentrations, tiller buds grew faster in OX lines than in WT, but they grew slower in Ri lines and mutant osnpf7.2. These results indicated that altered expression of OsNPF7.2 plays a significant role in the control of tiller bud growth and regulation of tillering. Elevated expression of OsNPF7.2 also improved root length, root number, fresh weight, and dry weight. However, reduced expression of OsNPF7.2 had the opposite result on these characters. OsNPF7.2 OX lines showed more significantly enhanced influx of nitrate and had a higher nitrate concentration than WT. The levels of gene transcripts related to cytokinin pathway and cell cycle in tiller bud, and cytokinins concentration in tiller basal portion were higher in OX lines than that in WT, suggesting that altered expression of OsNPF7.2 controlled tiller bud growth and root development by regulating cytokinins content and cell cycle in plant cells. Altered expression of OsNPF7.2 also was responsible for the change in expression of the genes involved in strigolactone pathway, such as D27, D17, D10, Os900, Os1400, D14, D3, and OsFC1. Conclusion Our results suggested that OsNPF7.2 is a positive regulator of nitrate influx and concentration, and that it also regulates cell division in tiller bud and alters expression of genes involved in cytokinin and strigolactone pathways, resulting in the control over rice tiller number. Since elevated expression of OsNPF7.2 is capable of improving rice grain yield, this gene might be applied to high-yield rice breeding. Electronic supplementary material The online version of this article (10.1186/s12284-018-0205-6) contains supplementary material, which is available to authorized users.

The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low-affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day-night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down-regulation of OsPTR9 in a T-DNA insertion line (osptr9) and in OsPTR9-RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.