The Rice ‘Nutrition Response and Root Growth’ (NRR) Gene Regulates Heading Date

Abstract

Dear Editor, As one of the most important developmental traits, flowering time has been extensively investigated in the long-day plant Arabidopsis. Multiple factors are involved, including photoperiod and/or temperature (Amasino, 2010Amasino R Seasonal and developmental timing of flowering.Plant J. 2010; 61: 1001-1013Crossref PubMed Scopus (615) Google Scholar). Flowering locus T (FT) is known to be an important integrator of different flowering pathways (Taoka et al., 2011Taoka K. Ohki I. Tsuji H. Furuita K. Hayashi K. Yanase T. Yamaguchi M. Nakashima C. Purwestri Y.A. Tamaki S et al.14–3–3 proteins act as intracellular receptors for rice Hd3a florigen.Nature. 2011; 476: U332-U397Crossref PubMed Scopus (451) Google Scholar). Genes of the CONSTANS (CO) family also play key roles in regulating flowering time. The CO protein consists of a zinc finger and a CCT domain shared by a group of plant-specific transcription factors that regulate photoperiodic flowering response and circadian rhythms in Arabidopsis. In the case of short-day plants such as rice, previous studies have also revealed a number of genes that control heading date, such as the CO ortholog Heading date 1 (Hd1) and, at its downstream, Heading date 3a (Hd3a), an ortholog of FT (Kojima et al., 2002Kojima S. Takahashi Y. Kobayashi Y. Monna L. Sasaki T. Araki T. Yano M Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions.Plant and Cell Physiology. 2002; 43: 1096-1105Crossref PubMed Scopus (849) Google Scholar; Komiya et al., 2008Komiya R. Ikegami A. Tamaki S. Yokoi S. Shimamoto K Hd3a and RFT1 are essential for flowering in rice.Development. 2008; 135: 767-774Crossref PubMed Scopus (417) Google Scholar). Although the main photoperiodic flowering regulators are highly conserved between Arabidopsis and rice, there are genes that act differently relating to the day-length property of rice, such as Early heading date1 (Ehd1), Indeterminate1 (OsID1), the MADS-box gene OsMADS51 (Doi et al., 2004Doi K. Izawa T. Fuse T. Yamanouchi U. Kubo T. Shimatani Z. Yano M. Yoshimura A Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-Iike gene expression independently of Hd1l.Gene Dev. 2004; 18: 926-936Crossref PubMed Scopus (638) Google Scholar; Kim et al., 2007Kim S.L. Lee S.Y. Kim H.J. Nam H.G. An G.H OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a.Plant Physiol. 2007; 145: 1484-1494Crossref PubMed Scopus (175) Google Scholar; Park et al., 2008Park S.J. Kim S.L. Lee S. Je B.I. Piao H.L. Park S.H. Kim C.M. Ryu C.H. Park S.H. Xuan Y.H. et al.Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod.Plant J. 2008; 56: 1018-1029Crossref PubMed Scopus (128) Google Scholar), and Ghd7 (Xue et al., 2008Xue W.Y. Xing Y.Z. Weng X.Y. Zhao Y. Tang W.J. Wang L. Zhou H.J. Yu S.B. Xu C.G. Li X.H. et al.Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice.Nat. Genet. 2008; 40: 761-767Crossref PubMed Scopus (1110) Google Scholar). Macronutrients are important factors to affect plant growth and development. To date, only a few studies of how nutrients affect plant flowering in Arabidopsis have been reported. The flowering time of the Arabidopsis phosphate deficiency mutant pho1 is delayed by 6 d compared with that of the wild-type plant (Poirier et al., 1991Poirier Y. Thoma S. Somerville C. Schiefelbein J A mutant of Arabidopsis deficient in xylem loading of phosphate.Plant Physiol. 1991; 97: 1087-1093Crossref PubMed Scopus (348) Google Scholar). The mutant insensitive to low phosphorus, lpi (low-phosphorus insensitive), flowers 3–5 d earlier than the wild-type plant (Sanchez-Calderon et al., 2006Sanchez-Calderon L. Lopez-Bucio J. Chacon-Lopez A. Gutierrez-Ortega A. Hernandez-Abreu E. Herrera-Estrella L Characterization of low phosphorus insensitive mutants reveals a crosstalk between low phosphorus-induced determinate root development and the activation of genes involved in the adaptation of Arabidopsis to phosphorus deficiency.Plant Physiol. 2006; 140: 879-889Crossref PubMed Scopus (145) Google Scholar), and nitrate reductase is found to play a role in regulating the flowering process by affecting the endogenous content of nitric oxide (Seligman et al., 2008Seligman K. Saviani E.E. Oliveira H.C. Pinto-Maglio C.A.F. Salgado I Floral transition and nitric oxide emission during flower development in Arabidopsis thaliana is affected in nitrate reductase-deficient plants.Plant and Cell Physiology. 2008; 49: 1112-1121Crossref PubMed Scopus (92) Google Scholar). Study on the function of Arabidopsis MYB62 provided information on its role in flowering, root development, and adaptive mechanisms during nutrient stress (Devaiah et al., 2009Devaiah B.N. Madhuvanthi R. Karthikeyan A.S. Raghothama K.G Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis.Mol. Plant. 2009; 2: 43-58Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Up to now, the molecular mechanism underlining the relationship between the nutrient application and plant reproductive growth is not clear. Our previous studies have demonstrated that NRR is alternatively spliced, producing two 5’-coterminal transcripts, NRRa and NRRb, both of which negatively modulate the rice root growth under the macronutrient-deficient conditions. NRRa is physically different from NRRb in that it possesses an 85-amino acid C-terminal extension harboring a CCT domain (Zhang et al., 2012Zhang Y.M. Yan Y.S. Wang L.N. Yang K. Xiao N. Liu Y.F. Fu Y.P. Sun Z.X. Fang R.X. et al.A novel rice gene, NRR responds to macronutrient deficiency and regulates root growth.Mol. Plant. 2012; 5: 63-72Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In order to find the functional difference between NRRa and NRRb, their overexpression and RNAi transgenic rice plants were analyzed intensively (see Supplemental Methods and Table 1). Confessedly, CCT motif functions as a nuclear localization signal. In fact, when transiently expressed as a GFP fusion protein in onion epidermal cells, NRRa was predominantly localized in the nucleus, whereas the NRRb–GFP fusion protein was mainly localized on the cell membrane (Figure 1A–1E). In order to verify the innate subcellular location of NRR in rice, transgenic rice plants expressing the NRR–eGFP fusion proteins driven by its native promoter (PNRR:NRRa–eGFP and PNRR:NRRb–eGFP) were produced (Supplemental Figure 1). The obvious nucleus localization was only observed in PNRR:NRRa–eGFP seedling root cells, other than PNRR:NRRb–eGFP transgenic rice (Supplemental Figure 2). These results suggested that NRRa containing the additional CCT domain would play some regulatory roles different from NRRb and, furthermore, the PNRR:NRRa–eGFP transgenic rice appeared significantly late flowering both in Beijing and in Hainan (Figure 1F); the expression level of NRRa was related to the time of delay in the heading date (Supplemental Table 2). But no difference was observed between the PNRR:NRRb–eGFP plants and vector-transformed control plants. These results showed that only expression of NRRa, not NRRb, affects the rice heading date. To understand the roles of NRRa and NRRb in regulating rice reproductive growth, the flowering of transgenic rice plants overexpressing NRRa, NRRb, or the RNA fragment encoding the CCT domain, as well as NRR RNAi plants (Zhang et al., 2012Zhang Y.M. Yan Y.S. Wang L.N. Yang K. Xiao N. Liu Y.F. Fu Y.P. Sun Z.X. Fang R.X. et al.A novel rice gene, NRR responds to macronutrient deficiency and regulates root growth.Mol. Plant. 2012; 5: 63-72Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), were intensively analyzed. Compared with wild-type plants, transgenic rice plants overexpressing NRRa driven by the CaMV35S promoter (35S:NRRa) exhibited a marked delay in the heading date (Figure 1G). In the tropical climate of the Hainan Province of China, wild-type plants flowered at about 85 d after sowing, whereas 35S:NRRa plants flowered at 97 d. In Beijing, wild-type plants flowered at around 116 d after sowing, whereas 35S:NRRa plants flowered between 133 and 143 d. Consistently with the late-flowering phenotype observed in plants overexpressing NRRa, the knockdown plants (NRRab–RNAi) flowered approximately 3 d earlier than the wild-type plants (Figure 1H). However, no obvious change in the heading date was observed in transgenic plants overexpressing NRRb or CCT. We also observed that the delayed heading date phenotype was restored in 35S:NRRa homozygous plants. To understand this phenomenon, we compared the expression levels of NRRa in heterozygous and homozygous NRRa plants segregated from the same line. The NRRa level in heterozygous plants was 30–50-fold higher than that of the vector-transformed control plants (Figure 1I and Supplemental Figure 3) but, in the homozygous 35S:NRRa plants, the level of NRRa decreased to the level close to that of wild-type plants. This may have led to the restored phenotypes in homozygous plants. The reason why the transgene is silent in the homozygous plants remains to be investigated. The influence of overexpression of NRRa on the expression levels of several rice flowering-related genes was examined in young leaf tissue of 60-day-old plants. Compared with wild-type plants, 35S:NRRa transgenic plants showed a sharp decrease in the expression of Hd3a and RFT1 and an increased level of the Ehd1 transcript (Figure 1J). The expression of Hd3a, RFT1, and Ehd1 in the tiller buds also showed similar expression patterns to that in the young leaf (Supplemental Figure 4). Taken together, we found that NRRa, but not NRRb, was involved in rice heading date regulation. Overexpression of NRRa in rice caused a substantial delay in the heading date, which was very likely mediated by direct or indirect suppression of two floral activator genes, RFT1 and Hd3a, and an increased expression of gene Ehd1. These results suggest that NRR can play multiple regulatory roles in the coordination of plant growth and development by means of alternative splicing, and understanding its roles in the nutrient–response pathway and the flowering pathway may help to reveal the interlinking of these two pathways. Supplementary Data are available at Molecular Plant Online.