Narrow albino leaf 1 is allelic to CHR729, regulates leaf morphogenesis and development by affecting auxin metabolism in rice

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1 DOI 1.17/s ORIGINAL PAPER Narrow albino leaf 1 is allelic to CHR729, regulates leaf morphogenesis and development by affecting auxin metabolism in rice Jing Xu 1,2 Li Wang 2 Mengyu Zhou 2 Dali Zeng 2 Jiang Hu 2 Li Zhu 2 Deyong Ren 2 Guojun Dong 2 Zhenyu Gao 2 Longbiao Guo 2 Qian Qian 2 Wenzhong Zhang 1 Guangheng Zhang 2 Received: 1 September 216 / Accepted: 5 January 217 / Published online: 2 February 217 Springer Science+Business Media Dordrecht 217 Abstract The leaf is the main site of photosynthesis and an important component of the ideotype in rice. Its development and morphogenesis directly affects rice yield. The rice mutant, narrow albino leaf 1 (), was obtained from a rice mutant population generated from an EMS-induced indica variety Shuhui527 (), and is mainly characterized by reduced plant height, narrow and albino leaves, a reduced number of crown roots, and an increased number of trichomes. Map-based cloning and genetic complementation experiments indicate that NAAL1, which encodes a CHR4/MI-2-like protein, is allelic with CHR729/OsCHR4, a gene known to regulate the methylation of H3K4 and H3K27. NAAL1 was expressed in a constitutive form in the rice root, stem, leaf sheath, and panicle. Further functional studies showed that NAAL1 had a Jing Xu, and Li Wang have contributed equally to this work. Electronic supplementary material The online version of this article (doi:1.17/s ) contains supplementary material, which is available to authorized users. Qian Qian qianqian188@hotmail.com Wenzhong Zhang zwzhong@126.com Guangheng Zhang zhangguangheng@126.com 1 Rice Research Institute of Shenyang Agricultural University / Key Laboratory of Northern Japonica Rice Genetics and Breeding, Ministry of Education and Liaoning Province / Key Laboratory of Northeast Rice Biology and Genetics and Breeding, Ministry of Agriculture, Shenyang 11866, Liaoning, China 2 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 316, Zhejiang, China combined effect with a plurality of genes that work within a network regulating leaf shape Moreover, this gene was involved in the synthesis, transport, and signaling of auxin, and thereby affecting the development of the root system, leaf vascular bundles, leaf morphogenesis, as well as the development of panicle traits. Keywords Rice (Oryza sativa L.) Leaf morphogenesis Auxin metabolism Pleiotropism Introduction Leaf morphology is part of the super-rice ideotype and its morphogenesis directly affects photosynthetic activity and rice yield (Yuan 1997). The leaf morphology is closely relat ed to plant spatial extension, effective leaf area for photosynthesis, photosynthetic efficiency, and dry matter accumulation. Leaf morphogenesis is often subject to the coordinated regulation of genetic inheritance, hormonal signaling, as well as environmental factors (Huang 23). The structure of cuticle, number and structure of vascular bundles, development of abaxial sclerenchyma, number of mesophyll cells, and size and number of vesicular cells may lead to changes in the polarity of a leaf. The development of the leaf polarity determines leaf length, leaf width, curling of the leaf tip, and other key indicators of rice leaf morphogenesis. In recent years, scientists have studied the regulatory mechanism for leaf morphogenesis at the molecular level, isolating and cloning many of the regulatory genes related to this regulation; among these genes, abaxially curled leaf 1 (ACL1) (Li et al. 21), rice outermost cell-specific (ROC5) (Zou et al. 211), rolling leaf14 (RL14) (Fang et al. 212), Zn-finger transcription factor (OsZHD1) (Xu et al. 214), SEMI-ROLLED LEAF1 Vol.:( )

2 176 (SRL1) (Xiang et al. 212), shallot-like 2 (SLL2) (Zhang et al. 215), R2R3-MYB transcription factor (OsMYB13L) (Yang et al. 214), and rolled and erect leaf 1 (REL1) (Chen et al. 215) lead to the inward or outward rolling of leaves by regulating the number, size, and distribution of bulliform cells on the adaxial surface. SHALLOT-LIKE1 (SLL1) (Zhang et al. 29) and semi-rolled leaf2 (SRL2) (Liu et al. 216) promote adaxial development of the abaxial leaf surface and propel the inward rolling of the leaf by regulating the differentiation of abaxial sclerenchyma. Curly flag leaf1 (CFL1) (Wu et al. 211) affects leaf rolling by regulating the development of cuticle. ADAXIALIZED LEAF1 (ADL1) (Hibara et al. 29) affects the adaxial development of epithelial cells and mesophyll tissues of the abaxial surface, which differentiates into alveolar cells and instigates leaf rolling to the abaxial leaf surface. OsAGO7 (Shi et al. 27) regulates leaf rolling by affecting the development of the adaxial/abaxial surface. The leaf width is directly associated with the number of leaf veins. When the leaf is narrow, the number of large and small leaf veins is reduced. However, when the leaf is wide, the number of leaf veins increases accordingly. Among the six cloned narrow leaf genes, including NARROW LEAF 7 (NAL7) (Fujino et al. 28), NARROW LEAF 1 (NAL1) (Qi et al. 28; Zhang et al. 214), NARROW AND ROLLED LEAF 1 (NRL1) (Hu et al. 21), NARROW LEAF 2/NARROW LEAF 3 nal2/3 (Cho et al. 213) and RICE MINUTE-LIKE1 (RML1) (Zheng et al. 216), the first two regulated leaf width by affecting the biosynthesis and polar transport of auxin respectively, while the last three by influencing the number, shape, and size of the vascular tissues simultaneously. Leaf development could be regulated by several groups of genes, mainly including YABBY genes (Dai et al. 27), auxin synthesis related YUCCA genes (Yamamoto et al. 27), and AUXIN RESPONSE FACTOR (ARF) (Tiwari et al. 23). These genes was predominantly expressed in the primordium differentiation period and seeding development (Toriba et al. 27; Cheng et al. 27). In rice, the YABBY family affects the growth of lateral organs. Notably, OsYAB3 (Dai et al. 27), OsYAB4 (Liu et al. 27) and DROOPING LEAF (DL) (Nagasawa et al. 23; Ohmori et al. 211) are associated with leaf development in rice, of which OsYAB4 is important for the vascular tissues development and DL for the development of the leaf midrib, sheath, and floral organs. Zhao et al. (21) has shown that YUCCA is rate limiting in auxin synthesis in plants, thereby playing an important role in the biosynthetic pathway of the tryptophan-dependent IAA. The expression of its family member OsYUCCA1 is limited to the tip of leaves, roots, and vascular tissues (Yamamoto et al. 27). ARF regulates the response to auxin and the development of the abaxial leaf surface (Waller et al. 22; Wang et al. 27). In addition, the rice gene FISH BONE (FIB) encodes a tryptophan amino transferase playing a key role in auxin synthesis in rice (Yoshikawa et al. 214), and PIN1 acts as an auxincarrier (Xu et al. 25), plays a major role in auxin-dependent adventitious root development and tillering, and it is expressed in the vascular tissues and primordial root. The narrow-albino leaf 1 () is a single recessive gene mutant that was obtained from mutant population generated from the ethyl methane sulfonate (EMS)-induced mutagenesis of the rice indica variety,. In contrast to the wild-type, the mutant was characterized as dwarfed plants mainly with narrow and albino upper leaves, a reduced shoot-to-root ratio, an increase in the number of trichomes. Map-based cloning results of the mutant identified the regulatory gene named NARROW ALBINO LEAF 1 (NAAL1) that is allelic to the reported gene CHR729/OsCHR4 that encodes a CHR4/MI-2-like protein (Hu et al. 212; Zhao et al. 212; Ma et al. 215). In this study, the function of NAAL1 was studied and the roles of NAAL1 in rice leaf morphogenesis, metabolic auxin regulation, and the rice yield formation were examined. Materials and methods Plant materials The mutant was identified from a rice mutant population generated by EMS-induced mutagenesis of the indica variety. The mutant was crossed with Nipponbare, and 1178 mutant plants were identified for gene mapping. All plants were cultivated in the experimental fields at the China National Rice Research Institute, either in Hangzhou, Zhejiang or Lingshui, Hainan during the normal growing season. Measurement of leaf traits and chlorophyll content The width and length of the upper three leaves of six plants in both the wild-type and the mutant were measured at the heading stage and averaged for data analysis. Root traits were investigated using roots analysis system (WSEEN LA-S Company, China) in both the wild type and the mutant. Other important agronomic traits, including plant height, panicle length, primary branch number per panicle, secondary branch number per panicle, number of grains per panicle, seed setting rate, 1-grain weight, grain length, and grain width, were measured according to the Standard Evaluation System for Rice ( knowledgebank.irri.org/ses/). Fresh leaves of three independent plants were sampled from the wild type and the mutant at the tillering stage, and were collected and chlorophyll content was measured. The leaf veins were excised and leaves were cut

3 into small pieces. Approximately.2 g of leaf tissue was placed into 25 ml of extraction solution containing 95% acetone and absolute ethanol (2:1, v/v). Extraction samples were incubated in the dark for 24 h at 28 C, the optical density (OD) at 663, 645, and 47 nm were measured using a DU8 spectrophotometer (Beckman Coulter, Germany). The methods used for pretreatment and chlorophyll determination were as previously described by Yang et al. (216). Scanning electron microscopy Leaves were taken from and wild-type plants at the heading stage and placed in 2.5% glutaraldehyde for more than 4 h, and rinsed three times with phosphate buffer and fixed in 1% OsO 4 in phosphate buffer at 4 C. Samples were washed in phosphate buffer for 15 min each time, then dehydrated through gradient ethanol solutions each step for 15 min. After that, samples were incubated in an ethanolisoamyl (1:1) acetate mixture for 3 min and transferred to isoamyl acetate for 1 h. Dried the samples with liquid CO 2, and coated the samples with gold palladium, finally examined using a Tabletop Microscope (Hitachi TM-1, Japan). Transmission electron microscopy Flag leaves taken from the mutant and wild type plants at the heading stage were placed in 2.5% glutaraldehyde and were placed under a vacuum until the leaves sank. Leaf samples were treated according to the method described by Zhao et al. (215) and analyzed with a transmission electron microscope (Hitachi H-765, Japan). Histology and microscopy The flag leaf samples were collected from the mutant and the wild type at the heading stage, and fixed the samples in 5% FAA overnight at 4 C. After that, the samples were dehydrated using an ethanol gradient, infiltrated with dimethylbenzene, and embedded in paraffin and then sectioned into 1 12 μm slices, stained with 1% safranine and 1% Fast Green, dehydrated using ethanol and infiltrated with dimethylbenzene again. Finally, a microscope (NIKON ECLIPSE 9I, Japan) was used for examination of the cellular microstructure in the leaf samples (Ren et al. 215). Map based cloning An F 2 population of 4913 plants was derived from a cross between the mutant and Nipponbare plants. A total of 1178 plants with mutant phenotypes were selected 177 for primary mapping by using simple sequence repeats (SSR) markers. For the target region, six sequence-tagged site (STS) markers were designed from the differences sequences between Oryza sativa L. ssp. japonica cultivar Nipponbare and indica cultivar obtained from the National Center for Biotechnology Information ( The molecular markers used are listed in Table S1. Construction of vectors and plant transformation For functional complementation, a vector was constructed by placing a NAAL1 full-length cdna sequence driven by its native promoter that was amplified from the wild type plant. This construct was introduced into the mutant by Agrobacterium tumefaciens-mediated transformation. The method for the vector construction was described by Zhao et al. (212). The primers for the complementation vector are listed in Table S1. To determine the subcellular localization of NAAL1, the coding sequence of NAAL1 without the stop codon was amplified and then inserted into the pcambia13-egfp vector to generate the 35S:NAAL1:GFP fusion construct. We then transformed the protein fusion construct and control vector into rice leaf protoplasts. The GFP signal was detected using an OLYMPUS IX71 confocal microscope. The GFP vector primers are listed in Table S1. RNA extraction and expression analysis To examine the expression pattern of NAAL1, we extracted RNA of leaves, roots, stems, sheaths, and panicles from the wild type using Axyprep Multisources Total RNA Miniprep Kit (USA-based Axygen Scientific, Inc). The RNA from leaves in both and were extracted from plants treated with 1-naphthaleneacetic acid (NAA). The RNA was reverse transcribed into cdna using Rever- Tra Ace qpcr RT Master Mix with gdna Remover Kit (TOYOBO, JAPAN). The amplification program for SYBR Green (95 C, 1s; 6 C, 3s; 72 C, 15s, for 4 cycles) was performed on an Applied Biosystems 79 HT Fast Real-Time PCR System (USA) following the manufacturer s instructions. Three replicates from each cdna sample were used for Real-Time PCR. Each sample was normalized to the amount of Actin transcript detected in the same sample. The RT-PCR primers used are listed in Table S1. NAA treatment Seeds of the mutant and the wildtype were germinated in a 1/2 MS liquid culture medium and were subjected to an NAA gradient treatment with six different concentrations of exogenous auxin ( nm,.1 nm, 1 nm,

4 178 1 nm,.1 µm, and 1 µm). The phenotypes were observed after 7 days of growth. The shoot length, maximum root length, and the root number of three independent plants in different concentrations of exogenous auxin were measured. The total root length was measured using the WSEEN LA-S Plant Roots Analysis System software. The relative expression of NAAL1 in the mutant and wild type seedlings grown in different NAA treatments was analyzed. The root samples of three independent plants that were grown in the free NAA treatment cultures were collected at 7 days after sowing and used for examination of the endogenous IAA content at the Institute of Botany, Chinese Academy of Sciences (Beijing, China). Results Phenotypic characterization of In this study, the narrow-leaf albino mutant,, was successfully separated from a rice mutant population generated by EMS-induction of the indica variety. The mutant was characterized as a dwarfed plant with upper-surface (adaxial) albino leaves and normal lowersurface color (Fig. 1d). Beginning at the seedling stage, had a narrower and shorter leaf compared to that of wild-type (Fig. 1a, b, c). The phenotype observations and statistics showed that the average width of the upper three leaves of wild-type at the heading stage were 1.81, 1.61, and 1.48 cm, respectively. However, those of were.63,.62, and.52 cm, which indicated an appreciable reduction of 65.2, 61.5, and 65.5%, respectively (Fig. 1g). Additionally, at the heading stage, the average length of the upper three leaves of the mutant were significantly reduced by 12.15, 16.27, and 19. cm, respectively, when compared to (Fig. 1f). Another notable feature of the mutant was abnormal root growth, which was observed as a significant reduction in the number of crown roots and total root length (Fig. 1e). At the seedling stage 7 days after sowing, the number of crown roots in the wild type and were 8.63 and 4.5, respectively, and their total root lengths were 56.6 and cm, respectively, for which relative to the wild-type dropped by and 37.17%, respectively (Fig. 1i, j).this results suggest that the reduction of the total root lengths of might be derived from the a c b f g Leaf length (cm) Leaf width (cm) naal 1 d e h i j Plant height (cm) Root number Root length (cm) Fig. 1 Phenotypic characterization of. a Cross-sections through the middle of the mature flag leaves of wild-type (WT, Shuhui527, left) and (right) plants, bar 1 cm. b Morphological difference between WT (left) and (right) at the tillering stage, bar 2 cm. c Comparison of the flag leaf of WT (left) and (right), bar 5 cm. d Differences of leaf shape and leaf color in the adaxial-side between WT (left) and (right), bar 1 cm. e Root difference between WT (left) and (right). Bar 2 cm. f, g Comparison of the upper three leaves between WT and ; leaf length (f) and leaf width (g) at maturity. h Comparison of the plant height of WT and at maturity. i j Comparison of the upper three leaves between WT and in crown root number (i), and total root length (j) in 1-day old seedlings. P <.5, P <.1; significance based on t-test

5 decrease of the number of crown roots. At the mature stage, the plant height of was only 7.9% of that in (Fig. 1h). Compared to, the mature panicle traits of, including panicle length, number of secondary branches per panicle, the number of spikelets per panicle, seed setting rate, and 1-grain weight, dropped sharply; the number of primary branches per panicle and the grain length also markedly declined. However, no significant difference was found in the grain width (Table 1). NAAL1 not only affects leaf morphology and root system development, but also has regulatory roles in panicle traits and the development of some grain morphologies. NAAL1 affects the development of leaf trichomes, the vascular bundle, and chloroplasts To observe changes in the leaf surface of the mutant, the leaves of wild-type and mutant plants were analyzed by scanning electron microcopy. Our observations showed that the number of large and small trichomes of the adaxial and abaxial surfaces of leaves increased relative to wild type. The number of large and small trichomes of the adaxial surface was 2.96 and 3.68 times greater than that of the wild type, respectively (Fig. 2 a, c, p) whereas those on the abaxial surface was 2.58 and 1.98 times greater, respectively (Fig. 2b, d, o). To examine the causes of the albino leaf phenotype in, the chlorophyll content and chloroplast structure of wild-type and mutant leaves at the peak of the tillering stage were measured and observed by acetone tissue processing and transmission electron microscopy (TEM). The contents of chlorophyll a and b, and carotenoids in at the tillering stage were 3.1,.94, and.55, respectively; while those in the mutant were 2.21,.68, and.45, respectively. This indicated a significant reduction of key photosynthetic molecules by 26.6, 27.7, and 18.2%, respectively, in the mutant (Fig. 2r). Additionally, the chloroplast structure in and was observed by TEM showed fewer chloroplast 179 membranes on the adaxial surface as compared to (Fig. 2e, f, i, j). However, less apparent differences were detected on the abaxial leaf surface (Fig. 2g, h, k, l) which data suggests that the upper-albino leaf phenotype in is caused by changes in the chloroplast structure on the adaxial surface of the leaf. To understand the narrow leaf phenotype in, the numbers of main and secondary leaf veins were analyzed in wild-type and the mutant under a stereomicroscope and by the paraffin method. As observed under the stereomicroscope, the number of the main and secondary veins in was reduced by 27.1 and 63.2%, respectively, compared to (Fig. 2q). The leaf transections made from paraffin embedded leaves showed that the number of vascular bundles in leaves were reduced by approximately 2% compared to, and the number of mesophyll cells in the direct vicinity of the adaxial vesicular cells was also significantly decreased in the mutant (Fig. 2m, n). Map based cloning of NAAL1 To locate the regulatory genes related to leaf narrowing in the mutant, was straightbred and reciprocally crossed with the japonica rice variety Nipponbare. The F 1 -generation phenotype was identified and the separation percentage of the F 2 -generation plants was based on differences in leaf shape. A Chi square test results showed that a single recessive gene pair controls the mutant phenotype (P =.978). A total of 1178 individual mutants with the narrow-leaf phenotype were chosen from the F2 population that consisted of 4913 individual plants. Using genome-wide screening, the NAAL1 gene was located in the region flanked by the RM6835 and RM11 SSR markers on chromosome 7. Through further development of new markers and the use of map-based cloning, the gene was eventually targeted between the STS markers P5 and P6 on BAC P5E2 at approximately 47.1 kb (Fig. 3a). The Rice Genome Table 1 The panicle traits of and rice plants Trait Panicle length (cm) ± ±.61 Number of primary branches per panicle ± ±..58 Number of secondary branches per panicle ± ± 1.15 Number of spikelets per panicle ± ± 7.37 Seed setting rate (%) ± ± 8.46 Grain length (mm) 11.2 ± ±.26 Grain width (mm) 2.67 ± ±.8 1-Grain weight (g) , Represents a significant difference between wild type and mutant at the.5 level and.1 level based on the t-test

6 18 a c b 2μm d 2μm 2μm 2μm e f g h i j k l n naal 1 1μm Lg Trich Sm Trich.2µm p Lg Trich Sm Trich q r LVB SVB 4 Chlorophyll content (mg/g) 1μm.2µm Number of vascular bundles o Trichomes number of abaxial side m.2µm Trichomes number of adaxial side.2µm chla chlb car Fig. 2 The differences in leaf trichome number, the vascular bundles, and chloroplasts between wild type and the mutant. a b A scanning electron micrograph of the adaxial (a) and abaxial (b) surfaces of leaves from WT. c d A scanning electron micrograph of the adaxial (c) and abaxial (d) surfaces of leaves from. e f Transmission electron micrographs of the adaxial surfaces of leaves from WT (e) and (f). g h Transmission electron micrographs of the abaxial surfaces of leaves from WT (g) and (h). i j Larger images of E and F. k l Larger images of G and H. m n Cross-sections of flag leavesfrom WT (m) and (n). o p Comparison of the number of trichomes on the abaxial side (o), and the adaxial side (p) of WT and leaves (Lg Trich, large trichomes; Sm Trich, small trichomes). q Comparison of the number of vascular bundles between WT and (LVB, the large vascular bundle; SVB, the small vascular bundle). r Comparison of the chlorophyll content between WT and (chla, chlorophyll a; chlb, chlorophyll b; car, carotenoid). P <.5, P <.1; significance based on the t-test (WT, Shuhui527) Annotation Databases ( cgi-bin/gbrowse/rice/) predicted that four possible open reading frames (ORFs) were present within the interval that encodes an unknown protein, a CHR4/MI-2-like protein, a peptide N-asparagine amidase, or a MYB family transcription factor, respectively. Sequencing and comparison with the wild-type indicated that LOC_ Os7g3145 encodes the CHR4/MI-2-like protein, and that this gene was mutated on the 652nd base, creating a substitution from an A to a T in the mutant (Fig. 3b). The LOC_Os7g3145 is thought to be NAAL1 that controls the leaf width. The complementation vector that contained the wildtype gene LOC_Os7g3145 sequence and its native promoter fragment was constructed. Through agrobacteriummediated transformation, the mutant was transformed and the functional complementation was verified by the identification of the transgene. Nine independent lines were obtained from the transgenic progeny, which exhibited normal leaf morphology. Among these lines, two independent transgenic lines were grown in paddy field and observed for complementation of the mutant phenotypes (Fig. 3c). The leaf phenotype indicated that the leaf narrowing, upper-albino leaf, and other mutant defects characteristic of 13

7 181 a Markers CHR.7 BACs RM6835 P1 P3 P5P6 P4 P2 RM11 OJ1457_D7 OJ1197_D6 P5E2 (GFP) and driven by 35S promoter. It was then transformed into rice leaf protoplasts. Using an Olympus IX71 confocal microscope, the expression of the 35S:NAAL1:GFP construct was observed in the nucleus, which is indicative of a nucleoprotein (Fig. 4b). b c Recombinants(38) (2)(1)() (3) the mutation were complemented in these transgenic plants (Fig. 3d). This complementation test showed that LOC_Os7g3145 is the target gene NAAL1, which regulates leaf width. NAAL1 is also allelic to CHR729/OsCHR4 (Hu et al. 212; Zhao et al. 212; Ma et al. 215), which regulates the methylation of H3K4 and H3K27. Expression pattern and subcellular localization of the NAAL1 protein (3) (38) NAAL1 Candidate Region Gene ATG A T TAG d -com Shuhui527 -com-1, -2 Fig. 3 Map-based cloning of NAAL1. a Fine mapping of NAAL1 on chromosome 7. NAAL1 was mapped primarily to the region flanked by RM6835 and RM11 on chromosome 7, and then narrowed to a kb region between P5 and P6 on BAC P5E2. b Gene structure of NAAL1. A base substitution from A to T was sequenced in the fifth exon of the ORF of the mutant. c Phenotypes of two independent complementation lines in transgenic rice plants at the heading stage grown in the field. d Flag-leaf morphology of WT,, -complementation-line-1 (naal-com-1), and -complementation-line-2 (naal-com-2) To characterize the expression pattern of NAAL1, quantitative real-time PCR (qrt-pcr) was applied to determine the expression of NAAL1 in different tissues and organs of the wild-type. Results showed that NAAL1 was constitutively expressed in the rice root, stem, leaf sheath, and panicle; the expression in the root was the highest, whereas that in the panicle was the lowest (Fig. 4a). Meanwhile, to identify the specific localization of NAAL1 in the cell, the ORF of NAAL1, was fused to the green fluorescent protein (8) Altered transcription level of auxin and leaf development related genes in Leaf development is subject to the coordinated control of genes, for example, leaf development YABBY genes, auxin synthesis related genes such as YUCCA genes, auxin transport-associated genes, and other leaf development related genes. To better understand the regulatory mechanism of NAAL1 that controls leaf morphogenesis, the expression of genes related to leaf development was compared in young developing leaves of wild-type and mutant plants. The qrt-pcr results showed that compared with the wild-type, the expression of YAB1, YAB4, YAB7, YUC1, YUC2, YUC4, YUC7, YUC8, ARF2, ARF5, and PINB1 was significantly downregulated in, whereas that of PIN1B (Wang et al. 29) was markedly upregulated (Fig. 5a). Moreover, the relative expression of SLL1, NAL7, NRL1, and NAL1, which are cloned genes related to rice leaf narrowing, were also significantly downregulated in the mutant; compared to the wild type, the expression of NAL1 was decreased to 3.6% in the mutant, and NRL1 was declined to 54.1% in leaves (Fig. 5c). These results show that the narrow-leaf phenotype of may be related to the altered transcriptional activity of leaf development-related and auxin-related genes. Response to different exogenous NAA concentrations on development To verify that NAAL1 was associated with the auxin metabolic pathway, the expression of FIB (Yoshikawa et al. 214), a key gene for the auxin tryptophan metabolic pathway, was analyzed. Results showed that the expression of NAAL1 in the mutant was decreased to 12.5% of that in wild type, thereby indicating that mutant plants had defects in auxin synthesis in which resulted in plant growth and morphological abnormalities (Fig. 5a). Thus, we examined endogenous IAA content of total plants in and at the seedling stage and found significant differences (Fig. 5b). Content of endogenous IAA in was declined by 12.5% compared with. To test this hypothesis, the seeds of both wild-type and the mutant were sown on 1/2 MS culture medium and subjected to a NAA gradient treatment upon germination. The NAA treatments consisted of six different concentrations of exogenous auxin ( nm,.1 nm, 1 nm, 1 nm,.1 µm, and 1 µm NAA) and the plant

8 182 Fig. 4 Expression pattern of NAAL1 and the subcellular localization of the NAAL1 protein in WT. a Relative expression levels of NAAL1 in leaf, root, stem, sheath, and panicle. b Subcellular localization of the NAAL1 protein in normal rice leaf protoplasts a Relative expression level of OsNAAL leaf root stem sheath panicle b 35S::GFP 1um 35S::OsNAAL1::GFP 1um phenotypes were observed after 7 days of growth (Fig. 6a, b). Results showed that without exogenous NAA, the shoot length of was similar to that of, the total root length (including lateral roots) was 8.12 cm longer than that of, and the maximum root length was 2.44 cm shorter than that of. With increasing NAA concentrations, the shoot length, maximum root length, and total root length of decreased gradually. However, 1 nm NAA, the shoot length and total root length of increased by 19.7 and 41.9%, respectively, compared to those conditions free NAA (Fig. 6c, f). When the concentration of NAA increased to 1 µm, the growth of the roots and shoots of and were inhibited to the greatest extent (Fig. 6c f). Without exogenous NAA, the expression of NAAL1 in was twice that in the mutant (Fig. 6g). With.1 nm NAA, the expression of NAAL1 in was rapidly reduced by 64.6% to only one-third of the normal level. With increasing concentrations of exogenous NAA, the expression always remained 2 4% that of the normal level in wild type. Under all NAA conditions, the expression of NAAL1 in the mutant was higher than that of the wild-type. In, the expression in plants treated with 1 nm NAA was 1.87 times that of plants not treated with NAA. When the concentration of exogenous NAA increased to 1 µm, no significant difference was observed in the wild type. The shoot and root lengths measured in plants grown in different NAA concentrations were consistent with the variation in NAAL1 expression. When considering the wild type phenotype and the expression of NAAL1, combined with the response to exogenous auxin (NAA), the results showed that the metabolic equilibrium of endogenous auxin was disrupted and the normal expression of NAAL1 was inhibited, thereby affecting normal plant growth and development. By contrast, in the mutant, the mutation in NAAL1 affected the auxin metabolic pathway and caused a decline in the synthesis of endogenous auxin. With the addition of 1 nm NAA, the expression reached maximum, and the root system development and plant growth of the mutant was partly recovered. Thus, the NAAL1 gene is involved in the tryptophan biosynthesis pathway of auxin production, thereby affecting root development and leaf morphogenesis. Discussion In this study, we identified a narrow albino leaf mutant, and used map-based cloning to found the underlying gene and named NAAL1 which was allelic to reported gene CHR729/OsCHR4. NAAL1 affected the development and morphogenesis of leaf and root by involving in the synthesis, transport, and signaling of auxin. In addition, the effects of NAAL1 on the development of chloroplast

9 183 a Relative expression level WT NAAL1 FIB YUC1 YUC2 YUC3 YUC4 YUC5 YUC6 YUC7 YUC8 YUC9 ARF1 ARF2 ARF3 ARF4 ARF5 PIN1 PIN1B PIN1C PIN2 PIN3 YAB1 YAB2 YAB3 YAB4 YAB5 YAB6 b.6 c 1.2 Content of IAA (µg/g) Relative expression level SLL1 NAL7 NRL1 NAL1 Fig. 5 The mutant has altered expression levels of auxin- and leaf development-related genes, and content of endogenous IAA. The rice Actin gene was used as an internal positive control to normalize the expression level of each gene investigated. Total RNA was extracted from leaves in the seedling stage. a Quantitative real time PCR (qrt-pcr) analysis was performed to study the transcript levels of NAAL1 and auxin-related genes (FIB, YUC family, ARF family, PIN family and YAB family) in. b Content of endogenous IAA in and. c Transcription levels of four cloned narrow leaf genes in were normalized to their levels in WT. Each column represents the mean ± SD of three biological replicates. P <.5, P <.1; significance based on t-test and trichomes were also observed. The effects of NAAL1 function on gene expression important to plant morphogenesis varied at different gene loci. For example, Hu et al. (212) found that CHR729, which is a CHD3 protein with dual capabilities in regulating chromatin, not only recognizes and modulates the methylation of H3K4 and H3K27 that can inhibit tissue-specific genes, but it is also involved in the morphogenesis of multiple organs in plants. Zhao et al. (212) has shown that CHR729/OsCHR4 could play an important role in the development of chloroplast in the mesophyll cells on the adaxial leaf surface, and the mutation of this gene could induce albino features on the adaxial surface. Ma et al. (215) determined that CHR729 regulates seedling development via the gibberellin biosynthetic pathway. Therefore, NAAL1 may be involved in multiple regulatory pathways and play important roles in the morphogenesis of major tissues and organs including roots, leaves, and panicles, thus showing pleiotropic effects in rice. Different genetic networks could regulate the effects of NAAL1 on the development of different tissues and organs; and details on the specific molecular mechanism are still pending. Leaf development is also regulated by a variety of phytohormones, especially at the stage of leaf primordium differentiation, and the metabolism of auxin greatly affects leaf morphogenesis (Bar and Ori 215). Among the isolated regulatory genes responsible for leaf shape, many genes are associated with the auxin metabolic pathway, for example, NAL1, a regulatory gene that controls the polar transport of auxin, affects leaf width (Qi et al. 28; Zhang et al. 214). NAL7, a member of the YUCCA gene family, is involved in the biosynthesis of auxin and also regulates leaf width (Fujino et al. 28). In the present study, we show that the expression of some members of YUC (a gene family in the tryptophan-dependent biosynthetic pathways), ARF (auxin transporters), and PIN (auxin carriers) families change significantly in when compared to wild type. Moreover, the expression of FIB, a key gene related to the biosynthetic pathway of auxin (tryptophan), was decreased to 12.5% in when compared to the wild type. As for root development,

10 184 a 2cm b 2cm M.1nM 1nM 1nM.1uM 1uM M.1nM 1nM 1nM.1uM 1uM c Shoot length (cm) nM 1nM 1nM.1uM 1uM d Max root length (cm).1nm 1nM 1nM.1uM 1uM e Root number nM 1nM 1nM.1uM 1uM f Total root length (cm) nM 1nM 1nM.1uM g Relative expression NAAL nM 1nM 1nM.1uM 1uM Fig. 6 Response to exogenous NAA concentration in. a b Phenotypes of 7-day-old wild type (WT, Shuhui527) and plants treated with or without an NAA gradient. c f Effects of exogenous NAA treatment on shoot length (c), maximum root length (d), root number (e), and total root length (f). Mean and SD values were obtained from three biological replicates. g Relative mrna levels of NAAL1 in leaves from 7-day-old seedlings of WT and treated with NAA. Mean and SD values were obtained from three biological replicates the role the NAAL1 mutation played was also observed. The max root length in free NAA culture was longer in than in wild type. When application of NAA, low concentrations of NAA (an exogenous auxin), partially restored the root growth of the mutant and the expression of NAAL1 was the highest, while with the increasing of concentrations, the inhibition effects of NAA on both the mutant and the wild type were shown (Fig. 6). Similar results were found in mutant fib for rice root development (Yoshikawa et al. 214), which played a pivotal role in IAA biosynthesis in rice. In addition, in plants, the expression of some genes related to transport and signaling of auxin had significant differences with the wild type (Fig. 5). Thus, we speculate that NAAL1 takes

11 part in the transport, signaling, and metabolism of auxin since auxin controls rice leaf morphogenesis and root development by a complex genetic network. Rice leaf width is positively correlated with the number of large/small vascular bundles. The mutation of NAAL1 causes deficiencies in the morphogenesis of multiple organs of the rice plant, which was particularly evident in its leaf shape. The narrow-leaf phenotype of the mutant is mainly attributed to a reduction in the number of large/small vascular bundles. At the seedling stage, the expression of known regulatory genes related to the leaf shape was analyzed in, and the expression of the regulatory genes correlated to the leaf narrowing phenotype. For example, SLL1, NAL7, NRL1, and NAL1 were downregulated. In addition, the expression of some members of the YAB family that are related to leaf morphogenesis, namely, YAB1, YAB4, and YAB6, were downregulated markedly. These results show that their expression is correlated, which suggests that they may control leaf morphogenesis in a coordinated or related way. NAAL1 and these genes control the leaf shape and play an important synergistic role in the leaf shape regulatory network, and future work will focus on the specific molecular mechanism of these networks. Studies have shown that leaf morphology of the upper three leaves after heading is closely related to panicle development in rice; the sugar content that accumulates in the rice grain primarily comes from photosynthesis in the leaf after heading, and the rice yield is greatly affected by the photosynthetic capacity of the leaf (Yin et al. 1956; Yoshida et al. 1973; Yang et al. 1999; Guo et al. 215). We show that a mutation in NAAL1 not only inhibits the root development at the seedling stage, but also significantly reduces the length and width of the upper three leaves and the total leaf area index. As a result, fewer products of photosynthesis accumulated so that a variety of plant developmental morphologies are altered, ultimately leading to a reduction in plant growth and grain yield. An exploration of the cloning and functions of NAAL1 will be instrumental in further revealing the genetic coupling in the source-sink balance of rice at the molecular level, thereby providing genetic resources and a theoretical basis for molecular breeding of super high-yield ideotypes. Acknowledgements This work was supported by National Key Research and Development Program (216YFD1181) and grants from the National Natural Science Foundation of China ( and ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No conflict of interest is declared. References 185 Bar M, Ori N (215) Compound leaf development in model plant species. 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