Volume 5, Issue 1, Pages 63-72 (January 2012) A Novel Rice Gene, NRR Responds to Macronutrient Deficiency and Regulates Root Growth  Zhang Yu-Man , Yan Yong-Sheng , Wang Li-Na , Yang Kun , Xiao Na , Liu Yun-Feng , Fu Ya-Ping , Sun Zong-Xiu , Fang Rong-Xiang , Chen Xiao-Ying   Molecular Plant  Volume 5, Issue 1, Pages 63-72 (January 2012) DOI: 10.1093/mp/ssr066 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Two Alternatively Spliced Transcripts of NRR. (A) cDNA fragments corresponding to NRRa and NRRb transcripts are amplified from rice root total RNA by RT–PCR. Lane 1, NRRb; Lane 2, NRRa; Lane M, DNA standards. (B) Schematic diagram of the generation of NRRa and NRRb from the primary NRR transcript. Ex1, Ex2, Ex3, and Ex4 stand for individual exons and In1, In2, and In3 for introns. UTR represents the 3′ untranslated region. Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Expressing Pattern of NRR in Rice. (A) The relative expression levels of NRRa and NRRb in different tissues by real-time RT–PCR. Different tissues were harvested from 90-day-old Japonica wild-type plants. ACTIN was used as internal control. Error bars indicate ±SE (n = 3). (B) The X-Gluc-staining of different tissues of the NRR promoter:GUS transgenic rice (right); the tissues from Japonica wild-type plant were used as negative control (left). Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Transcription of NRR under Various Conditions of Nutrient Starvation as Detected by Real-Time RT–PCR. (A) and (B) were the relative transcription levels of NRRa and NRRb in rice seedling roots cultured in ½ MS (control) or NPK-deprived media. ½ MS is the NPK sufficient medium, used as control, while -NPK is a modified ½ MS in which the NPK nutrients are depleted. The 12-day-old rice seedlings cultured in ½ MS media were treated by NPK starvation or fresh ½ MS media for different time periods. The ‘re1hr’ columns represent the resupplying of NPK only to -NPK media. After 72 h of treatment, we just simply replaced both ½ MS and -NPK media with the fresh ½ MS media, and continued to culture for 1 h. At each time point, the expression of NRRa or NRRb in nutrient starvation was compared to that of the corresponding control. The transcription levels of NRRa and NRRb in NPK-deprived media for 72 h increased significantly compared to that in ½ MS media. (C) and (D) show the relative levels of NRRa and NRRb in rice seedling roots under long-term nutrient starvation treatments. The pre-germinated seeds were sown directly in ½ MS (control), N-, P-, K-, or NPK-deprived media for 7, 12, and 15 d, respectively. The gene expression levels under different nutrient stresses were compared to that of the control in the same day. The expression of both NRRa and NRRb exhibited statistically significant increased levels under N- and NPK-starvation. In (A–D), the ACTIN transcript was used as an internal control. The relative expression level resulted from three experimental replicates. Error bars indicate SE (n = 3). * and ** indicate the significant difference at P = 0.05 and 0.01, respectively, in SPSS paired-samples t-test. Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 GUS Expression Directed by the NRR Promoter in Rice Seedling Roots under Various Nutrition Conditions. NRR promoter:GUS transgenic rice seeds were cultured in ½ MS (CK), N-, P-, K- NP-, or NPK-deficient medium (-N, -P, -K, -NP, or -NPK) for 12 d, and the seedling roots were stained for GUS activity. GUS was weakly expressed at the base area of the primary root under nutrient-sufficient conditions (CK), but strongly expressed under conditions of N-, P-, and NP-starvation. Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Knock-Down of NRRa and NRRb Resulted in Enhanced Root Growth under Various Nutritional Conditions. (A) Expression levels of NRRa and NRRb in knock-down plants determined by semi-quantitative RT–PCR. NRRab–RNAi, transgenic plants in which both NRRa and NRRb were knocked down; miRNRRb and miRNRRa, transgenic plants in which expression of either NRRb or NRRa was specifically suppressed by artificial microRNA; CK, the pCAMBIA1300 vector-transformed plants; M, DNA standards. ACTIN was used as an internal control. (B) Root phenotypes of the 12-day-old rice seedlings cultured in ½ MS medium. All the NRRa- and/or NRRb-knock-down plants had primary and adventitious roots slightly longer than those of the wild-type Nipponbare plants (WT). (C) Root phenotypes under P-starvation. While root growth in the wild-type plant became inhibited, all the knock-down plants showed long primary and adventitious roots. Among them, the NRRa/b doubly repressed plants (NRRab–RNAi) displayed stronger root phenotypes compared with that of the single knock-down plants (miRNRRa or miRNRRb). (D) Root phenotypes under conditions of N-depletion. All the knock-down plants showed longer primary and adventitious roots than those of the wild-type plants. Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 The Inhibited Root Growth Phenotype in NRRa-OX Plants. (A) Expression levels of NRRa, NRRb, and CCT in transgenic seedling shoots as determined by semi-quantitative RT–PCR. Compared to wild-type seedlings (WT), high-level expression of NRRa, NRRb, and CCT was detected in the corresponding transgenic plants. ACTIN, and NRRb in the case of NRRa-OX plants, were used as the internal controls. (B–D) 25-day-old plants of NRRa-OX and wild-type. (B) demonstrates the conspicuous inhibited root growth in the NRRa-OX compared to the wild-type plants. (C) and (D) demonstrate the comparison of fresh root weight and root length in NRRa-OX and wild-type plants: NRRa-OX plants displayed significant decreases in these aspects. Error bars indicate SE (n = 7). * and ** indicate the significant difference at P = 0.05 and 0.01, respectively, in SPSS paired-samples t-test. (E) The comparison of the root growth of 68-day-old plants of wild-type (left) and NRRa-OX (right)—the roots of NRRa-OX plants exhibited inhibited growth compared to that of wild-type plants. Molecular Plant 2012 5, 63-72DOI: (10.1093/mp/ssr066) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions